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Cited by 34Bacterial Genetics and BiotechnologyMicrobial Metabolic Engineering and BioproductionProtein purification and stability

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

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Hausjell, Johanna, Kutscha, Regina, Gesson, Jeannine D., Reinisch, Daniela, Spadiut, Oliver·2020·Bioengineering
DOI 10.3390/bioengineering7010008PMC PMC7175309
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Abstract

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

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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 [74]

    Materials and Methods §2.1 (page unavailable) identifies the stated OmpA-leader sequence on both chains of a genome-integrated Fab construct. The Introduction identifies FabZ as the antigen-binding fragment used. The paper does not report comparing leader sequences or selecting OmpA as the best. (LLM, conf=0.86)

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Authors
Hausjell, J., Kutscha, R., Gesson, J. D., Reinisch, D., & Spadiut, O.
Affiliations
  • TU Wien (AT)
  • Boehringer Ingelheim (Austria) (AT)
  • TU Wien, Institute of Chemical, Environmental and Bioscience Engineering, Research Division Biochemical Engineering, 1060 Vienna, Austria
ORCID
Venue
Bioengineering
Publisher
MDPI AG
Published
2020-01-06
Volume
7
Issue
1
Pages
8
ISSN
2306-5354 (ISSN-L)
DOI
10.3390/bioengineering7010008
PMC
PMC7175309
OpenAlex
W2997456998
Cited by
34
References
41
Citations by year
2026: 2 · 2025: 5 · 2024: 4 · 2023: 4 · 2022: 1 · 2021: 13 · 2020: 5
FWCI
3.37
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioengineering (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Boehringer Ingelheim RCV GmbH &amp; Co KG (-)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
lactose induction, T7 expression system, Escherichia coli, lac repressor, lactose uptake, cell lysis, IPTG induction, recombinant protein expression, cell fitness

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