title: Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product.
abstract: Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0.47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q302 were attempted. Addition of Triton X-100, L-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q302 and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q302 was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q302 closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.
authors.affiliation: Green Cross Corporation, Osaka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tsujikawa
authors.forename: K
authors.initials: K
authors.lastname: Okabayashi
authors.forename: M
authors.initials: M
authors.lastname: Morita
authors.forename: T
authors.initials: T
authors.lastname: Tanabe
mesh_terms: D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D010710:Phosphates; C024788:ammonium phosphate; D001120:Arginine; D010450:Endopeptidases; D014568:Urokinase-Type Plasminogen Activator