ABSTRACT
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.
