Document Type
Article
Publication Date
11-1-2019
Publication Title
European Journal of Pharmaceutical Sciences
Abstract
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.
Recommended Citation
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.
https://engagedscholarship.csuohio.edu/scichem_facpub/525
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
DOI
10.1016/j.ejps.2019.105066
Version
Postprint
Publisher's Statement
Link to publisher version: https://doi.org/10.1016/j.ejps.2019.105066
Volume
139
Comments
This work was supported by grants from the NIH (1R15HL138544- 01, X.-L. Sun), National Science Foundation MRI Grant (CHE-1126384, X.-L. Sun) and Faculty Research Development Grant and the Research Fund from the Center for in Health and Disease (GRHD) at Cleveland State University. X. Liu appreciates the China Oversea Scholar Award from China Scholarship Council.