Anti-HER2 scFv expression in SHuffle host

Introduction and aim: Breast cancer is the leading cause of cancer death in women worldwide. Relationship between expression of human epidermal growth factor 2 (HER2) and malignity of breast tumors has been led to the production of monoclonal antibodies targeting HER2+ tumors. Single chain variable fragment (scFv) is the smallest functional region of antibody for antigen binding. scFvs improve their penetrability into tumors while retaining specific affinity and having low immunogenicity. Inparticular, anti-HER2 scFv can efficiently target the therapeutical proteins, drugs, and nanoparticles toward HER2+ cancer cells. In this study, a novel Escherichia coli protein expression strain, SHuffle, was used for expression of anti-HER2 scFv. SHuffle is trxB-, gor-, which helps disulfide bond formation in cytoplasm, and overexpresses cytoplasmic DsbC which plays chaperon role to enhance protein-folding capacity. This E. coli strain is an ideal host for expression of disulfide-bonded recombinant proteins Method and material: Anti HER2-scFv gene was cloned in expression vectors pET22 b (+) and pET28. Anti HER2-scFv expression was induced with 1 mM IPTG in the growth temperature of30°C. The expression of anti HER2-scFv in the SHuffle host was investigated by SDS-PAGE. Furthermore, the protein expression was confirmed using Western blot analysis probed with anti- His antibody.Result: The protein bands around 28 kDa were detected in the SDS-PAGE gel. Furthermore, Western blot analysis using anti-His antibody confirmed the successful expression of Anti HER2-scFv. Conclusion: The results showed that SHuffle is a suitable strain for the expression of anti HER2-scFv containing two disulfide bonds

Keywords:

Breast cancer , scFv , HER2 , E. coli SHuffle , Expression vectors