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Updated: May 1, 2026
Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells
Published on: May 9, 2025
Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in
Vajihe Akbari1, Hamid Mir Mohammad Sadeghi, Abbas Jafrian-Dehkordi
1Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Hezar Jarib Avenue, Isfahan, Iran.
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.
Insights
Researchers produced a HER2-specific single chain variable fragment (ScFv) in E. coli. This purified ScFv shows high purity and binds to HER2 receptors, indicating potential for targeting HER2-overexpressing cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The human epidermal growth factor receptor (HER) family is crucial for cell growth and signaling.
- Aberrant HER function is implicated in various cancers, making it a therapeutic target.
- Receptor dimerization is essential for HER signal transduction.
Purpose of the Study:
- To produce and purify a single chain variable fragment (ScFv) targeting domain II of the HER2 receptor.
- To evaluate the feasibility of using Escherichia coli for ScFv production.
- To assess the biological activity and potential therapeutic application of the anti-HER2 ScFv.
Main Methods:
- Expression of anti-HER2his-ScFv in E. coli BL21 (DE3) cytoplasm.
- Purification of ScFv using Ni-NTA affinity chromatography.
- Characterization of ScFv purity and molecular weight via SDS-PAGE and Western blotting.
- Assessment of biological activity through binding assays on HER2-positive BT-474 cells.
Main Results:
- The anti-HER2his-ScFv was successfully produced in E. coli, primarily as inclusion bodies.
- Purification yielded approximately 29 mg/l of ScFv with near 95% purity.
- SDS-PAGE and Western blotting confirmed the ScFv molecular weight at approximately 27 kDa.
- The purified ScFv demonstrated specific binding to HER2 receptors on BT-474 cells.
Conclusions:
- A HER2-specific ScFv was efficiently produced and purified from E. coli.
- The ScFv exhibits biological activity, binding to the HER2 receptor.
- This ScFv represents a promising candidate for developing targeted therapies against HER2-overexpressing tumors.
