Construction and characterization of a recombinant human beta defensin 2 fusion protein targeting the epidermal growth factor receptor: in vitro study
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.
KeywordsHER2/neu hBD2 Single-chain variable fragment Fusion protein Cancer cell targeting Cytotoxicity
This work was supported by grant of the Science Foundation of Wenzhou in China (No. Y20090279), grant of Guangdong Provincial Department of Science and Technology (No. 2009B080701090), the Program for New Century Excellent Talents in University (NCET-08-0611) and the Academy of Sciences comprehensive strategic cooperation of Guangdong Province in China (No. 2010B090301016).
Conflict of interest
The authors declare that they have no conflict of interest.
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