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Cited by 34Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityHER2/EGFR in Cancer Research

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli

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Akbari, Vajihe, Mir Mohammad Sadeghi, Hamid, Jafrian-Dehkordi, Abbas, Abedi, Daryoush, Chou, C Perry·2014·Journal of Industrial Microbiology and Biotechnology
DOI 10.1007/s10295-014-1437-0
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Abstract

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand–HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.

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  • Expression of an scFv against HER2 derived from Trastuzumab in SHuffle at 30 °C resulted in enhanced solubility and higher expression level compared to its expression in BL21(DE3) at 37 °C.

    “Recently, it has been reported that the expression of an scFv against HER2 derived from Trastuzumab (drug bank number DB00072) in SHuffle at 30 °C resulted in enhanced solubility and a higher expression level of the molecule as compared to its expression in BL21 (DE3) at 37 °C [109,110,111,112].”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [110]

    The Results and discussion (page unavailable) identify the scFv as derived from pertuzumab, not trastuzumab. The paper reports expression in BL21(DE3), with optimal production at 37 °C and the highest soluble-to-insoluble ratio at 25 °C; it does not report the claimed SHuffle-versus-BL21 comparison. (LLM, conf=0.90)

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  • The final production yield of anti-HER2 scFv was 147 mg/L under optimal expression conditions (24 h after induction with 0.05 mM IPTG at 30 °C, LB medium, SHuffle strain).

    “The final production yield of the anti-HER2 scFv was 147 mg/L, under optimal expression conditions (24 h after induction with 0.05 mM IPTG at 30 °C, LB medium, SHuffle strain).”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [110]

    The Results and discussion section states that optimal anti-HER2 scFv production occurred with 1 mM IPTG at 37 °C, contrary to the claimed 0.05 mM at 30 °C. The Bacterial strains, plasmid, and reagents section identifies BL21(DE3), not SHuffle, as the expression host and describes 2 hours of induction, not 24. The cited passages do not establish the claimed 147 mg/L yield; page numbers are unavailable in our copy. (LLM, conf=0.89)

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Authors
Akbari, V., Mir Mohammad Sadeghi, H., Jafrian-Dehkordi, A., Abedi, D., & Chou, C. P.
Affiliations
  • Isfahan University of Medical Sciences (IR)
  • grid.411036.1 000000011498685X Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Research Center, Faculty of Pharmacy Isfahan University of Medical Sciences Hezar Jarib Avenue Isfahan Iran
  • grid.46078.3d 0000000086441405 Department of Chemical Engineering University of Waterloo 200 University Avenue N2L 3G1 Waterloo ON Canada
Corresponding author
Abedi, Daryoush
ORCID
Akbari, Vajihe
Venue
Journal of Industrial Microbiology and Biotechnology
Publisher
Oxford University Press (OUP)
Published
2014-06-01
Volume
41
Issue
6
Pages
947-956
ISSN
  • 1367-5435 (ISSN-L)
  • 1476-5535
DOI
10.1007/s10295-014-1437-0
OpenAlex
W2038499837
Cited by
34
References
1
Citations by year
2026: 1 · 2025: 4 · 2024: 2 · 2023: 2 · 2022: 4 · 2021: 3 · 2020: 5 · 2019: 3 · 2018: 1 · 2017: 1 · 2016: 4 · 2015: 3 · 2014: 1
FWCI
2.35
Type
journal article
Language
en
Open access
Licence
academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
Locations
  • Journal of Industrial Microbiology & Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
Funders
Isfahan University of Medical Sciences (189113)
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
  • Health Sciences › Medicine › Oncology › HER2/EGFR in Cancer Research
Keywords
scFv, HER2, epidermal growth factor receptor 2, recombinant antibody production, Escherichia coli BL21(DE3), inclusion bodies, Ni-NTA affinity purification, BT474 cells, HER2-targeted therapy, monoclonal antibody alternatives
MeSH terms
Antineoplastic Agents, Escherichia coli, Humans, Receptor, ErbB-2, Erb-b2 Receptor Tyrosine Kinases, Cell Line, Tumor, Single-Chain Antibodies, Antibodies, Monoclonal, Humanized

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