Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity
Abstract
Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2 + breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle ® strain as it is trxB - , gor - , and overexpresses cytoplasmic DsbC chaperone. In this study, the anti-HER2 scFv was successfully expressed and purified in BL21 (DE3) and SHuffle ® cells. Here, significant higher soluble anti-HER2 scFv was produced in SHuffle ® than in BL21 strain. The specific binding of anti-HER2 scFv to HER2 was shown by flow cytometry analysis and ELISA. Moreover, it was demonstrated that the anti-HER2 scFv produced in SHuffle ® binds to HER2 at higher level as compared to that expressed in BL21 cells. Furthermore, competitive ELISA-based study suggested that anti-HER2 scFv recognizes the same epitope of HER2 receptor as the trastuzumab antibody. Our findings indicated that correct disulfide bond formation in SHuffle ® strain can result in enhanced solubility and higher biological activity level of anti-HER2 scFv.
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The anti-HER2 scFv yield with CyDisCo was 70.5% higher than that obtained in SHuffle using LB medium (147 mg/L).
“and 70.5% higher than that obtained in SHuffle using the LB medium (147 mg/L) [109].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [109]
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 [109]
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 [109]
Cites
Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli
Sørensen, H. P., & Mortensen, K. K. (2005) · Microbial Cell Factories · doi:10.1186/1475-2859-4-1 · Cited by 841 · see citing works
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Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments
Sandomenico, A., Sivaccumar, J. P., & Ruvo, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176324 · Cited by 134 · see citing works
“scFv pelB/pET22b 1 mM IPTG 25 ◦C 24 h BL21(DE3) LB pKJE7 DnaKdnaJ-grpE 65 ug/ml [109]”
BackgroundInfluential
- Authors
- Ahmadzadeh, M., Farshdari, F., Nematollahi, L., Behdani, M., & Mohit, E.
- Affiliations
- Shahid Beheshti University (IR)
- Pasteur Institute of Iran (IR)
- Corresponding author
- Mohit, Elham
- Venue
- Molecular Biotechnology
- Published
- 2019-11-05
- Volume
- 62
- Issue
- 1
- Pages
- 18-30
- ISSN
- 1073-6085 (ISSN-L)
- 1559-0305
- OpenAlex
- W2989112412
- Cited by
- 40
- References
- 1
- Citations by year
- 2026: 6 · 2025: 5 · 2024: 8 · 2023: 5 · 2022: 4 · 2021: 7 · 2020: 5
- FWCI
- 2.64
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Molecular Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Shahid Beheshti University of Medical Sciences
- 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-positive breast cancer, recombinant protein solubility, disulfide bond formation, inclusion bodies, epitope recognition, trastuzumab
- MeSH terms
- Trastuzumab, Antibodies, Monoclonal, Epitopes, Breast Neoplasms, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Female, Humans, Recombinant Proteins, Solubility, Gene Expression, Erb-b2 Receptor Tyrosine Kinases, Receptor, ErbB-2, Single-Chain Antibodies
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