title: Expression and functional characterization of a recombinant targeted toxin with an uPA cleavable linker in Pichia pastoris.
abstract: A recombinant targeted toxin (Disintegrin-Conj-Mel) was developed that contained a disintegrin connected to cytotoxic melittin by a urokinase plasminogen activator (uPA)-cleavable linker. This recombinant targeted toxin was designed to target tumor cells expressing integrin αvβ3. The fusion gene was expressed under the control of the promoter AOX1 in Pichia pastoris. Electrophoresis by SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain, demonstrated that an approximately 13 kDa fusion protein was secreted into the culture medium. The molecular weight was that calculated from the predicted amino acid sequence. After optimizing the growth and expression conditions of the transformant strain, about 160 mg/L of the recombinant protein was achieved. The recombinant protein was purified to more than 95% purity by SP Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The hemolysis bioactivity test revealed that the fusion had no hemolytic activity or cytotoxicity against uPA non-expressing 293 cells, but exerted dose-dependent inhibition on uPA-expressing A549 cell proliferation.
authors.affiliation: Department of Biomedicine, Institute of Frontier Medical Sciences, Jilin University, Changchun, Jilin, China.
authors.forename: Wen he
authors.initials: Wh
authors.lastname: Zhu
authors.forename: Miao nan
authors.initials: Mn
authors.lastname: Sun
authors.forename: Yong sheng
authors.initials: Ys
authors.lastname: Wang
authors.forename: De Jun
authors.initials: DJ
authors.lastname: Sun
authors.forename: Shao xuan
authors.initials: Sx
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods*; D003435:Crotalid Venoms / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D019483:Disintegrins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes; D006461:Hemolysis; D006801:Humans; D008555:Melitten / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D003435:Crotalid Venoms; D019483:Disintegrins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D008555:Melitten; C047981:arginyl-glycyl-aspartic acid; D014568:Urokinase-Type Plasminogen Activator