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.

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

“…AOX1-deleted transformants could grow slowly in the methanol medium because they had another alcohol oxidase encoded by the AOX2 gene, which is expressed more weakly than the AOX1 gene . The frequency of AOX1 disruption by double crossing-over (37%) was identical to the frequency reported for the production of hepatitis B antigen by Cregg et al (1987), tumour necrosis factor by Sreekrishna et al (1989) and human single-chain urokinase-type plasminogen activator (Tsujikawa et al, 1996).…”

Expression of Recombinant Proteins in Pichia Pastoris

“…The addition of Triton X-114 during rPIN-a (recombinant puroindoline-a) fermentation increased the production yield of the protein by 10-fold to 13 mg/l and inverted the ratio between secreted and membane-bound rPIN-a [46]. The addition of 0.01% (v/v) Triton X-100 to a feeding medium reduced partially the proteolysis of a urokinase-type plasminogen activator and increased the secretion level [47].…”

The yeast expression system for recombinant glycosyltransferases

“…For expression in P. pastoris there is no general rule concerning the need of N-glycosylation for efficient secretion. Indeed, secretion efficiency of a-Nacetylgalactosaminidase is strongly decreased when the Nglycosylation is missing [110], but Tsujikawa et al [111] reported secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site. The gal-T1 produced by P. pastoris is recognized directly by polyclonal antibodies and when analyzed on SDS-PAGE it comigrates with the human gal-T1 indicating that no hyperglycosylation takes place (Malissard et al unpublished results).…”