Purification And Characterization Of Anti-tumor Protein MAP30 From Bitter Melon

MAP30(Momordica Antiviral Protein 30 Kd),a single-stranded type-I ribosome inactivating protein which is isolated from bitter melon,possesses versatile biological activities,such as antivirus,antitumor,antimicrobial activities.The anti-tumor activity of MAP30 is of much concern and it has been considered to be a desirable anti-cancer drug candidate.However,the clinical effect of MAP30 on tumor is hampered by several factors,one of which is the cell membrane.Since the cell membrane hinders cell-impermeable molecules into cells,cell-penetrating peptides(CPPs)have been used as a powerful tool to deliver designated cargos across the plasma membrane.HBD,a novel CPP derived from the C-terminus of human extracellular superoxide dismutase,was previously found and demonstrated to own high cell-penetrating efficiency and low cell-toxicity.Therefore,we aimed to improve MAP30 penetrating ability and cell toxicity via introducing HBD.In this research,we reported a method to overcome the cell membrane barrier that MAP30 was modified with a cell-penetrating peptide(named HBD)by genetic recombination method.rMAP30 and rMAP30-HBD protein was mainly expressed in soluble form in E.coli by deleting its signal peptide sequence;By fusing His-tag at the C-terminal,rMAP30-HBD at high purity was obtained after Ni-NTA affinity chromatography purification;The supercoiled double-stranded DNA cleavage and cell-free translational assays demonstrated that rMAP30 retained its inherent biological activities and fusing with a cell penetrating peptide of HBD to MAP30 did not interfere with the activity of MAP30 itself;The cell uptake test,cell viability assay,Annexin V-FITC apoptosis experiment and in vivo studies using a xenograft tumor mouse model all demonstrated that HBD peptide significantly enhanced the penetrating ability and cytotoxicity of MAP30 on tested tumor cells.To our knowledge,this is the first report about MAP30 conjugated with a cell penetrating peptide which showed higher anti-tumor activity than MAP30 itself and extended the application of MAP30 in cancer therapy area.