European Journal of Pharmaceutical Sciences
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 (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 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 rTM456 modification. Recombinant TM456 with a C-terminal LPETG tag (rTM456-LPETG) was expressed in Escherichia coli for its end-point modification with NH2-diglycine-PEG5000-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific PEGylation of rTM456 via CFCC. Both PEGylated rTM456 conjugates retained protein C activation activity as that of rTM456. Also, they were more stable than rTM456 in Trypsin digestion assay. Further, both PEGylated rTM456 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.
Liu, Xia; Boron, Mallorie; Zhao, Yu; and Sun, Xue-Long, "End-point Modification of Recombinant Thrombomodulin with Enhanced Stability and Anticoagulant Activity" (2019). Chemistry Faculty Publications. 525.
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Link to publisher version: https://doi.org/10.1016/j.ejps.2019.105066
Available for download on Sunday, November 01, 2020