The bacterial plasminogen-activator staphylokinase (Sak) is a promising thrombolytic agent for treating acute myocardial infarction. To effectively reduce the immunogenicity of Sak while maintaining its fibrinolytic activity, site-specific PEGylation was performed in the present study. The chemoselective cysteine PEGylation site was selected within an immunodominant region (amino acid residues 71-87) using an in silico approach. The PEGylated Sak variants prepared in this study showed a purity of >97.0%. PEGylation at Position 80 resulted in a Sak variant Sak(E80C-PEG) which has similar fibrinolytic activity and thermostability compared with the native recombinant staphylokinase (r-Sak). The immunogenicity of Sak(E80C-PEG) in guinea pigs was greatly reduced compared with the native r-Sak. Furthermore, preliminary pharmacokinetic results suggested that the plasma clearance of Sak(E80C-PEG) from the blood stream of rabbit was significantly decreased compared with that of r-Sak, resulting in a 2.8-fold increase of initial half-life and a 3.8-fold increase of systemic availability. In summary, these results demonstrated that site-specific PEGylation yielded a novel Sak variant Sak(E80C-PEG) with remarkable advantages over the unmodified Sak.
Keywords: fibrinolytic activity; immunogenicity; pharmacokinetic; site-specific pEGylation; staphylokinase.
© The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.