Site-specific PEGylation of a truncated thrombomodulin mutant at the C terminus via the Staudinger reaction resulted in unchanged enzymatic activity.
Site-specific PEGylation of a truncated thrombomodulin mutant at the C-terminus successfully retains full enzymatic activity, offering a strategy to improve protein stability without loss of function.
Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)-like domains 4-6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme.
Cazalis et al. (Sat,) reported a other. Site-specific PEGylation at the C terminus vs. Un-PEGylated thrombomodulin construct was evaluated on Enzymatic activity. Site-specific PEGylation of a truncated thrombomodulin mutant at the C terminus via the Staudinger reaction resulted in unchanged enzymatic activity.