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Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. The 4, 5, 6 EGF-like domain of TM (TM) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM is a promising anticoagulant candidate but has a very short half-life. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM modification. Recombinant TM with a C-terminal LPETG tag (rTM-LPETG) was expressed in Escherichia coli for its end-point modification with NH-diglycine-PEG-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM-LPETG via SML with NH-diglycine-PEG-azide, which facilitates a site-specific PEGylation of rTMvia CFCC. Both PEGylated rTM conjugates retained protein C activation activity as that of rTM. Also, they were more stable than rTM in Trypsin digestion assay. Further, both PEGylated rTM conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.
This article was published in the following journal.
Name: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences
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