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Published on: October 15, 2016
Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity
Maryam Ahmadzadeh1,2, Farzaneh Farshdari1, Leila Nematollahi3
1Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, No. 2660, Vali-e-Asr Ave, Tehran, 1991953381, Iran.
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.
Insights
The SHuffle® strain enhances the production of soluble anti-HER2 single-chain variable fragments (scFv) for breast cancer therapy. This improved expression leads to higher biological activity and specific binding to HER2-positive cancer cells.
Area of Science:
- Biotechnology
- Oncology
- Molecular Biology
Background:
- Breast cancer is a leading global cancer, with HER2-overexpressing subtypes associated with poor prognosis.
- HER2-targeting monoclonal antibodies improve survival, but single-chain variable fragments (scFv) offer better tumor penetration.
- Bacterial expression of scFv can be challenging due to disulfide bond formation and inclusion body production.
Purpose of the Study:
- To evaluate the efficacy of the SHuffle® bacterial strain for expressing soluble anti-HER2 scFv.
- To compare the solubility, binding affinity, and biological activity of anti-HER2 scFv produced in SHuffle® versus BL21 (DE3) strains.
- To confirm the specific targeting of HER2 by the expressed scFv.
Main Methods:
- Expression and purification of anti-HER2 scFv in BL21 (DE3) and SHuffle® bacterial strains.
- Solubility assessment of purified scFv.
- Flow cytometry and ELISA to confirm specific binding to HER2.
- Competitive ELISA to compare epitope recognition with trastuzumab.
Main Results:
- Significantly higher soluble anti-HER2 scFv was produced in the SHuffle® strain compared to BL21.
- Anti-HER2 scFv expressed in SHuffle® demonstrated enhanced binding to HER2.
- Competitive ELISA indicated that the scFv recognizes the same HER2 epitope as trastuzumab.
Conclusions:
- The SHuffle® strain facilitates proper disulfide bond formation, enhancing scFv solubility and biological activity.
- This optimized expression system provides a more effective anti-HER2 scFv for potential therapeutic applications.
- The study highlights the importance of bacterial strain selection for recombinant protein production in cancer research.
