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 influence of the carbohydrate moiety on the stability and functional characteristics 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 signal codons of SK with those from alpha-factor leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P. pastoris after its integration into the host chromosome. Western blot and zymographic analysis of proteins secreted from the recombinant P. pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly, corresponding to a 55-kDa protein product as compared to the 47-kDa native SK. The glycosylated SK retained a plasminogen activation capability identical to that of its unglycosylated counterpart. Glycoform SK exhibited an enhanced stability profile at 25 degrees C and 37 degrees C and improved resistance towards 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 glycosylation of SK results in 30-40% enhancement of the protein stability and resistance towards degradation but does not interfere with its fibrinolytic function.
Pratap
J
J
Institute of Microbial Technology, Chandigarh, India.
Rajamohan
G
G
Dikshit
K L
KL
D000595:Amino Acid Sequence; D006031:Glycosylation; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010957:Plasmids; D010960:Plasminogen Activators / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D013300:Streptokinase; D010960:Plasminogen Activators