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.

• Authors: Maryam Ahmadzadeh, Farzaneh Farshdari, Leila Nematollahi, Mahdi Behdani, Elham Mohit
• Journal: Molecular biotechnology (Mol Biotechnol) Vol. 62 Issue 1 Pg. 18-30 (Jan 2020) ISSN: 1559-0305 [Electronic] United States
• PMID: 31691197 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Epitopes
• Recombinant Proteins
• Single-Chain Antibodies
• ERBB2 protein, human
• Receptor, ErbB-2
• Trastuzumab

Topics

• Antibodies, Monoclonal (metabolism, therapeutic use)
• Breast Neoplasms (metabolism)
• Enzyme-Linked Immunosorbent Assay
• Epitopes (immunology)
• Escherichia coli (genetics, metabolism)
• Female
• Gene Expression (immunology)
• Humans
• Receptor, ErbB-2 (immunology)
• Recombinant Proteins (metabolism)
• Single-Chain Antibodies (genetics, immunology, isolation & purification)
• Solubility
• Trastuzumab (metabolism)