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

Human single-chain urokinase-type plasminogen activator without an N-g lycosylation site (scu-PA-Q(302)) was produced in the methylotrophic y east, Pichia pastoris using the shortened prepeptide sequence of a fun gal aspartic proteinase, Mucor pusillus rennin (MPR). The level of uro kinase-type plasminogen activator (u-PA) immunoreactive material in YP M 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 tw o-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA- Q(302) were attempted. Addition of Triton X-100, L-arginine and ammoni um phosphate to the YPM medium minimized the proteolysis of scu-PA-Q(3 02) and increased the yield of immunoreactive material to approximatel y 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of ye ast did not reduce the degradation. Go-expression of scu-PA-Q(302) and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q(302) purified from the culture supern atant of the improved medium by two successive chromatographies on Phe nyl-Sepharose and S-Sepharose. The purified protein had a molecular we ight of 47 kDa. It did not contain detectable N-linked oligosaccharide s, 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-Q(302) closely resembles natural scu-PA wi th respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.