Construction and characterization of a recombinant human beta defensin 2 fusion protein targeting the epidermal growth factor receptor: in vitro study

• Published in: Applied microbiology and biotechnology 2013-05, Vol.97 (9), p.3913-3923
• Main Authors: Zhang, Minjing, Qiu, Zhuangwei, Li, Yinyu, Yang, Yan, Zhang, Qihao, Xiang, Qi, Su, Zhijian, Huang, Yadong
• Format: Article
• Language: English
• Subjects: Adaptive immunity; Antibodies; Antigens; Apoptosis - drug effects; beta-Defensins - genetics; beta-Defensins - metabolism; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Breast cancer; Cell Line, Tumor; Chemical properties; Chromatography; Chromatography, Affinity; Cloning; Cytotoxicity; Drug resistance; E coli; Electron microscopes; Epidermal growth factor; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Genetic aspects; Genetic recombination; Glycoproteins; Humans; Kinases; Leukemia; Life Sciences; Lymphoma; Medical research; Microbial Genetics and Genomics; Microbiology; Peptides; Physiological aspects; Plasmids; Proteins; Receptor, Epidermal Growth Factor - immunology; Receptor, Epidermal Growth Factor - metabolism; Receptor, ErbB-2 - immunology; Receptor, ErbB-2 - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Recombinant proteins; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism; Studies
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-012-4257-z

The HER2/neu proto-oncogene encodes a 185-kDa trans-membrane glycoprotein kinase with extensive homology to the epidermal growth factor receptor and plays a key role in the transformation and growth of malignant tumors. To date, two antibody drugs targeting HER2/neu have been developed successfully. In order to reduce the cost and the time of clinical treatment, we produced a fusion protein composed of human beta defensin 2 (hBD2) and anti-HER2/neu single-chain variable fragment (scFv 4D5), which is capable of specifically targeting, significantly inhibiting, and promptly killing HER2/neu-positive cancer cells. The recombinant protein was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal expression level reached to 40.2 % of the total supernatant protein. After purifying by Ni-NTA affinity chromatography, the fusion protein was cleaved with a SUMO-specific protease to obtain hBD2–4D5, which was further purified by Ni-NTA affinity chromatography. The purity of hBD2–4D5 was higher than 95 %, and the yield was 19 ± 2 mg/L in flask fermentation. The cell number count and flow cytometry results showed that hBD2–4D5 exerted cytotoxic and anti-proliferative effects on HER2/neu-positive breast cancer cell line, SKBR-3. The results of scanning electron microscope and transmission electron microscope observation indicated that hBD2–4D5 could induce intracellular ultrastructure changes and cell necrosis by disrupting the cell membrane. Immunofluorescence analysis showed that hBD2–4D5 could bind to SKBR-3 cells and further be internalized into the cytoplasm. Moreover, hBD2–4D5 could also mediate apoptosis of SKBR-3 cells by up-regulating the ratio of Bax to Bcl-2.