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.
Institute of Biomedicine, Jinan University, Guangzhou 510632, China.
Minjing
M
Zhang
Zhuangwei
Z
Qiu
Yinyu
Y
Li
Yan
Y
Yang
Qihao
Q
Zhang
Qi
Q
Xiang
Zhijian
Z
Su
Yadong
Y
Huang
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D066246:ErbB Receptors / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism; D023083:beta-Defensins / Q000235:genetics / Q000378:metabolism*
D066246:ErbB Receptors; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D023083:beta-Defensins; C106641:DEFB4A protein, human; C000715470:MAS1 protein, human