Expression，Purification And Activity In Vitro Of Human β Defensin2Targeting HER2

OBJECTIVE:Recombinant protein HBD2-4D5which fused with scFv4D5and human betadefensin2(HBD2) was expressed in Escherichia coli Origami B (DE3) by smallmolecule ubiquitin-like modifier protein (SUMO). To explore the mechanism ofHBD2-4D5on breast cancer cells in vitro, we determined its anti-tumor activity andspecific binding; this mechanism provides an initial basis for the study of targetingpeptide.METHODS:The cDNA fragments coded the his-tagged SUMO-4D5and SUMO-HBD2-4D5wassubcloned into the vector pET-3c to construct the recombinant expression vectorspET3c-SUMO-HBD2-4D5and pET3c-SUMO-4D5, and then the plasmids weretransformed into Origami B (DE3). Recombinant proteins SUMO-4D5andSUMO-HBD2-4D5were purified by Ni-NTA Sepharose FF, Sephadex G-25and SUMOprotease from the supernatants of bacteria lysis. Specific binding capacity of4D5andHBD2-4D5were analyzed by Immunofluorescence laser confocal scanning microscopy.CCK-8was used to detect HBD2,4D5and HBD2-4D5cytotoxicity of breast cancer cells,and we used flow cytometry to detect breast cancer cell apoptosis in4D5and HBD2-4D5,and analyzed by Western Blot. Scanning electron microscopy and transmission electronmicroscopy detect HBD2,4D5and HBD2-4D5on breast cancer cell ultrastructure.Results:Under the optimal expression conditions, the fusion proteins HBD2-4D5and4D5were detected in the soluble fraction of transformed Origami B (DE3). The fusion proteinSUMO-HBD2-4D5and SUMO-4D5were purified. The final yield of HBD2-4D5was25mg per liter, while4D5was40mg per liter. Immunofluorescence-based internalization analysis shown both HBD2-4D5and4D5were localized primarily in the cytoplasm ofSK-BR-3cells after treatment, but not in MDA-MB-231cells. Similar phenomenon wasobserved in HER2/neu-positive SK-OV-3cells. Compared to cells treated by4D5orHBD2in the same condition, HBD2-4D5treatment of HER2/neu-positive SK-BR-3cellsresulted in marked inhibition of growth in dose dependant manner, and the IC50value ofHBD2-4D5against the SK-BR-3cells was3.2±0.17μM, but low expression of HER2inthe fusion protein of the same molar concentration of HBD2-4D5human breast cancercells MDA-MB-231did not show significant inhibition. Control protein HBD24D5in thesame molar concentration of SK-BR-3, MDA-MB-231had no significant inhibitory effect.However, for HER2/neu-negative MDA-MB-231cells, no obvious morphologicalchanges and minor cytotoxicity of HBD2-4D5were observed in the same dosage thattreated in SK-BR-3cells. Flow cytometry analysis showed that HER2/neu-negativeMDA-MB-231cells were treated with HBD2-4D5and4D5only induced little partialcells apoptosis (15.65±2.32%vs.13.16±2.11%) even in the highest tested dose (6.4μM),which was in accordance with the cytotoxicity assay result in MDA-MB-231cells.HBD2-4D5caused32.6±3.32%and67.15±5.13%of HER2/neu-positive SK-BR-3cellsapoptosis in response to3.2μM and6.4μM, which was far more effective than the sametreatment of4D5on SK-BR-3cells. Western blot analysis showed that the SK-BR-3weretreated by3.2μM HBD2-4D5for24h, the Bax/Bcl-2increased300%; MDA-MB-231under the same conditions that Bax and Bcl-2expression than the normal group had nosignificant changes. Scanning electron microscopy and transmission electron microscopy(TEM) test results revealed that HBD2-4D5target role in HER2-positive cell membraneand attack the membrane, which showed obvious features of apoptosis.Conclusion:Fusion protein HBD2-scFv4D5，based on the anti-HER2which toward HER2/neupositive cells, dose to cause membrane rupture and apoptosis play the role of its kill cells.