Research On Preparation And Pharmaceutical Properties Of Site-Specifically PEGylated Staphylokinase

PEGylation can effectively improve the protein efficacy by prolonging serum half-life and reducing proteolytic sensitivity and immunogenicity. Staphylokinase (SAK), whose substrate is plasminogen, belonging to biological macromolecules, is a novel thrombolysis agent for therapy of myocardial infarction. The clinical defects of SAK, such as short circulating half-life and high immunogenicity, can be overcome effectively by PEGylation. However, due to the steric hindrance of polyethylene glycol (PEG), PEGylation may decrease the bioactivity of proteins by interfering with the interaction of proteins and their substrates, especially for those proteins which possess high molecular weight substrates. In the present study, we altered the PEG conformation and steric shielding effect through the linker between PEG and SAK in order to find a method that can improve the bioactivity of PEGylated proteins.Site-specific PEGylation at the site far from the bioactive domain of proteins is a feasible approach to decrease the effects on bioactivity of PEGylated proteins. Therefore, the recombinant SAK, used in our research, was inserted with Cys residue at C-terminus far away from the bioactivity domain. PEG-20K was covalently conjugated to the free thiol in Cys residue of SAK by three bifunctional reagents respectively. The three monoPEGylated SAKs had the same structure except for the linkers (i.e., the phenyl, propyl, and amyl moieties). The phenyl moiety was a rigid linker, whereas the propyl and the amyl moieties represented short and long flexible linkers respectively. Circular dichroism and fluorescence spectra indicated that the protein structure of the three PEGylated SAKs was not altered visibly as compared to that of the native SAK. Dynamic light scattering and analytical ultracentrifugation detection displayed that rigid phenyl linker induced dense PEG conformation that could extensively shield most domains adjacent to C-terminus while the flexible propyl and amyl linkers led to loose conformation that could wrap extensive surface of SAK. As compared with loose PEG conformation, dense PEG conformation had lower steric shielding effect on SAK. As to pharmaceutical properties, dense PEG conformation was more efficient to increase the plasma half-life, decrease the proteolytic sensitivity and immunogenicity as well as enhance the in vitro bioactivity (by 15%) of the PEGylated SAK than loose PEG conformation.In addition, we prepared six PEGylated SAKs in order to systematically investigate the effects of the PEG length, the PEGylation site and linker chemistry on the bioactivity of the heat-treated PEGylated SAK. Circular dichroism and fluorescence spectra showed that PEGylation at site far away from bioactive domain with high molecular weight PEG and flexible linker could more effectively maintain structure of SAK during heat treatment at 70℃ for 2 h. Analytical ultracentrifugation detection revealed that heat treatment could change loose PEG conformation to dense one, thereby increasing the relative bioactivity of C-terminally PEGylated SAK with 20 kDa PEG and amyl linker by 27%. Conclusively, site-specific PEGylation with dense PEG conformation had lower PEG steric hindrance and could improve the bioactivity of PEGylated protein. The present strategy may rationally optimize and tailor the PEGylation of proteins possessing high molecular weight substrates.