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Cited by 26Protein purification and stabilityMonoclonal and Polyclonal Antibodies ResearchBacterial Genetics and Biotechnology

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

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Sina, Mohammad, Farajzadeh, Davoud, Dastmalchi, Siavoush·2015·Advanced Pharmaceutical Bulletin
DOI 10.15171/apb.2015.062PMC PMC4729350
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Abstract

PURPOSE: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. METHODS: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. RESULTS: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. CONCLUSION: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein.

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  • Use of fusion partners such as GST, MBP, SUMO and Trx to target proteins, especially scFv fragments, results in solubility-enhancing properties and increased yields of soluble and active products.

    “Several reports have also suggested that the use of fusion partners such as GST [118], maltose-binding protein (MBP) [119,120], small ubiquitin-related modifier (SUMO) [87,121] and thioredoxin (Trx) [122] to the target proteins, especially to scFv fragments, results in solubility-enhancing properties and in increased yields of soluble and active products.”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [118]

    The citation [122] is attached specifically to thioredoxin (Trx), not to the other fusion partners. The Introduction describes a GST-fused scFv, and the Discussion and Conclusion attribute changes in its soluble expression to cultivation conditions; the passages found for thioredoxin and Trx do not address whether a Trx fusion increases soluble, active product yields. (LLM, conf=0.76)

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Cited by

Sorted by citations

Authors
Sina, M., Farajzadeh, D., & Dastmalchi, S.
Affiliations
Tabriz University of Medical Sciences (IR)
Corresponding author
Dastmalchi, Siavoush
ORCID
Venue
Advanced Pharmaceutical Bulletin
Publisher
Maad Rayan Publishing Company
Published
2015-11-30
Volume
5
Issue
4
Pages
455-461
ISSN
  • 2228-5881 (ISSN-L)
  • 2251-7308
DOI
10.15171/apb.2015.062
PMC
PMC4729350
OpenAlex
W2212632382
Cited by
26
References
1
Citations by year
2026: 1 · 2025: 1 · 2023: 1 · 2022: 6 · 2021: 3 · 2020: 4 · 2019: 3 · 2018: 3 · 2017: 3 · 2016: 1
FWCI
1.01
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Advanced Pharmaceutical Bulletin (publishedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Institutional Research Information System (Università degli Studi di Brescia) (submittedVersion, other-oa)
Funders
  • Tabriz University of Medical Sciences
  • University of Tabriz
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
humanized monoclonal antibody, Escherichia coli expression, inclusion bodies, lactose induction, IPTG, low-temperature cultivation, osmotic shock, magnesium chloride, sucrose, sorbitol, recombinant protein solubility

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