Expression, Purification And Activity Analysis Of SUMO-HBD2-4D5 Fusion Protein In Escherichia Coli

OBJECTIVE:A Prokaaryotic recombinant vector expressed fusion protein SUMO-HBD-4D5 was constructed and transformed into E. coli. The target proteins were purified and their biological activities of antibacterial and anti-cancer was analyzed.METHODS:The cDNA fragments coded SUMO and HBD2-4D5 were amplified by PCR respectively and inserted into vector pET-3c to construct the recombiant expression vectors. Recombinant proteins, HBD2-4D5 was purified using the combination of Ni-NTA Sepharose FF, digestion by specific SUMO protease and Sephadex G-25.The antibacterial activity of HBD2-4D5 was detected by staphylococcus aureus (ATCC25923) and E.coli K12D31. The effects of proliferation inhibition on SK-BR-3 and MDA-MB-231 cells were measured by MTT assay and flow cytometry assay. Binding affinity was analyzed by immunofluoresence assay.Results:The target proteins were high-level expressed in E.coli Origami B (DE3). HBD2-4D5 was purified using the combination of Ni-NTA Sepharose FF, digestion by specific SUMO protease and Sephadex G-25, and the purity was higher than 95%. The target protein was confirmed by Western bolt and MALDI-TOF. The efficacy of proliferation inhibition activity was assayed by MTT on on SK-BR-3 and MDA-MB-231 cells showed that HBD2-4D5 had proliferation inhibition activity to SK-BR-3 cells and the IC50 is 103.325μg/ml. The target protein HBD2-4D5 showed significant antibacterial activity against E.coli K12D31 and staphylococcus aureus (ATCC25923). Also, HBD2-4D5 demonstrated selectivingly binding activity to SK-BR-3, a HER2 over expressed breast cancer cell line.Conclusion:SUMO-HBD2-4D5 was high-level expressed and purified successfully.In In vitro assay of HBD2-4D5 shows significant antibacterial activity and selectivingly binding activity to HER2 recepter.