Molecular Cloning of DARPins G3 in pET28b Expression Vector and Optimization of the Expression of This Protein in Escherichia Coli

Background: Human epidermal growth factor receptor 2 (HER2) is over-expressed in breast, ovarian, gastric, and prostate cancers used as a tumor marker the diagnosis of cancer. Monoclonal antibodies have been diagnostic therapeutic tool against HER2. Because difficulties associated with stability complexity construct high cost antibody production, we aimed to investigate, cloning, expression HER2- binding DARPins genes identify, HER2-positive markers, investigate. Materials Methods: After synthesis, peptide gene was cloned into M13 vector sub-cloned TOP10 pet28b bacterial vector. culturing bacteria on an agarose plate containing antibiotics, transfected expressing were selected. To ensure enzymatic digestion recombinant plasmid delivery for sequencing. Isopropyl β-d-1-thiogalactopyranosideIPTG induction protein SDS-PAGE method Western blot confirmation. Results: The polymerase chain reaction (PCR) amplification product analyzed using gel electrophoresis. Plasmid purified from positive clone by PCR sequenced cloning confirmed. competent cells, obtained colonies. SDS- PAGE results showed effect different conditions including temperature, IPTG concentration, time pET-DARPins expression. Conclusion: We succeeded express new codon-optimized Escherichia coli HEK293t system.