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End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity.

08:00 EDT 9th September 2019 | BioPortfolio

Summary of "End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity."

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.

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This article was published in the following journal.

Name: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences
ISSN: 1879-0720
Pages: 105066

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A vitamin-K dependent zymogen present in the blood, which, upon activation by thrombin and thrombomodulin exerts anticoagulant properties by inactivating factors Va and VIIIa at the rate-limiting steps of thrombin formation.

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