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Cited by 21Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityBacterial Genetics and Biotechnology

A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

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Petrus, Marloes L. C., Kiefer, Lukas A., Puri, Pranav, Heemskerk, Evert, Seaman, Michael S., Barouch, Dan H., Arias, Sagrario, van Wezel, Gilles P. +1 more·2019·Applied Microbiology and Biotechnology
DOI 10.1007/s00253-019-10145-1PMC PMC6851033
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Abstract

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

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  • Using the pSAR2 plasmid with a rhamnose promoter, the scFv PGT135 antibody fragment was successfully produced in the periplasmic space, with amounts up to about 1.2 g/L of biologically active scFv PGT135 recovered.

    “In particular, by using the pSAR2 plasmid with a rhamnose promoter, the scFv PGT135 antibody fragment was successfully produced in the periplasmic space. Amounts up to about 1.2 g/L of the biologically active scFv PGT135 were recovered [71].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [71]

    The Results and Discussion report that the rhamnose-inducible pSAR-2 vector produced PGT135 scFv directed to the periplasm, and that the purified fragment was biologically active. But the approximately 1.2 g/L figure (1182 ± 2 mg/L) is the amount measured in total cells in shaker flasks, not the amount of active antibody recovered: about 0.5 g/L was soluble, and the Discussion reports about 58 mg/L purified. (LLM, conf=0.93)

    Experimental — accuracy not yet measured

  • Petrus and coworkers reported a robust scalable expression system for production of scFvs in the periplasmic space based on the pSAR-2 vector, obtaining HIV-neutralizing PGT135 scFv with a yield of 1.2 g/L after 48 h induction at 25 °C using 15 mM l-rhamnose in shake flasks.

    “The HIV-neutralizing PGT135 scFv antibody fragment was obtained with an outstanding yield of 1.2 g/L after 48 h of induction at 25 °C using 15 mM l-rhamnose in shake flasks [71].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [71]

  • Expression of an scFv antibody fragment using the novel vector pSAR-2 containing the pelB leader sequence and the rhamnose-inducible expression promoter obtained a yield of 1.2 g/L.

    “Recently, the expression of an scFv antibody fragment has been used as a showcase for validating the efficacy of the novel vector pSAR-2 containing the pelB leader sequence and the rhamnose-inducible expression promoter, obtaining a yield of 1.2 g/L [71].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [71]

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Authors
Petrus, M. L. C., Kiefer, L. A., Puri, P., Heemskerk, E., Seaman, M. S., Barouch, D. H., Arias, S., van Wezel, G. P., & Havenga, M.
Affiliations
  • Leiden University (NL)
  • Batavia Biosciences (Netherlands) (NL)
  • Beth Israel Deaconess Medical Center (US)
Corresponding author
Havenga, Menzo
ORCID
Venue
Applied Microbiology and Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2019-10-22
Volume
103
Issue
21-22
Pages
8875-8888
ISSN
  • 0175-7598 (ISSN-L)
  • 1432-0614
DOI
10.1007/s00253-019-10145-1
PMC
PMC6851033
OpenAlex
W2981899766
Cited by
21
References
4
Citations by year
2026: 4 · 2025: 6 · 2024: 2 · 2022: 4 · 2021: 2 · 2020: 3
FWCI
1.49
Type
journal article
Language
en
Open access
Licence
Locations
  • Applied Microbiology and Biotechnology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Leiden Repository (Leiden University) (submittedVersion, cc-by)
  • Data Archiving and Networked Services (DANS) (submittedVersion, cc-by)
Funders
Leiden University
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 › Genetics › Bacterial Genetics and Biotechnology
Keywords
scFv antibody fragments, HIV neutralizing antibodies, Escherichia coli expression, superfolder GFP, inclusion bodies, batch bioreactor, HIV prevention, monoclonal antibodies
MeSH terms
Antibodies, Monoclonal, Escherichia coli, Promoter Regions, Genetic, HIV Antibodies, HIV-1, HIV Infections, Gene Expression, Bioreactors, Single-Chain Antibodies, Antibodies, Neutralizing

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