Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation

Degradation of streptokinase (SK) has been frequently observed during large -scale protein production. An enhanced susceptibility of SK to degradation has been correlated with its existence in a partially unfolded state. The i nfluence of the carbohydrate moiety on the stability and functional charact eristics of SK has been examined by obtaining the glycoform of SK following its secretion through the methylotrophic yeast Pichia pastoris. Secretion of the protein product was achieved by replacing the native secretion signa l codons of SK with those from a-factor leader peptide and expressing the f usion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosom e. Western blot and zymographic analysis of proteins secreted from the reco mbinant P. pastoris indicated that SK was glycosylated by the host cells, w hich resulted in the appearance of a SK species migrating slowly, correspon ding to a 55-kDa protein product as compared to the 47-kDa native SK. The g lycosylated SK retained a plasminogen activation capability identical to th at of its unglycosylated counterpart. Glycoform SK exhibited an enhanced st ability profile at 25 degrees C and 37 degrees C and improved resistance to wards protease treatment compared to unglycosylated SK secreted through P. pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli. The results presented thus illustrate that N-linked glyco sylation of SK results in 30-40% enhancement of the protein stability and r esistance towards degradation but does not interfere with its fibrinolytic function.