Thrombomodulin (TM), an endothelial cell surface membrane glycoprotein, is a cofactor for protein C activation via thrombin, and therefore a crucial regulator in protein C pathway and coagulation cascade. TM contains six epidermal growth factor (EGF)�like structures, in which the forth to sixth EGF-like region (TM456) is the minimum functional domain responsible for the activity of protein C activation. Therefore, TM456 is attractive anticoagulant drug candidate. Herein, we report site-specific functionalization of recombinant thrombomodulin (rTM) with poly(ethylene glycol) (PEG) and lipid via sortase A (SrtA)-mediated ligation (SML) and copper-free click chemistry (CFCC). SrtA is a transpeptidase from S. aureus that recognizes and cleaves LPXTG motif on the target protein, and then catalyzes the linkage of the target protein with the compound containing glycine. CFCC is a bioorthogonal reaction of dibenzocyclooctyne group (DBCO) with azide linker to form a stable triazole. A truncated rTM456 derivative with azidohomoalanine for CFCC modification and LPETG tag at the C-terminal for the recognition of SrtA was expressed in E. coli. and purified with Nickel affinity column. Site-specific PEGylation and lipidation of rTM456 were performed by SML as well as CFCC. The conjugation products of PEGylation and lipidation were confirmed by SDS-PAGE and western blot. Site-specific functionalizations of rTM leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity and pharmacokinetic properties.

Site-specific functionalizations of recombinant thrombomodulin by enzymatic and chemical methods

246th National Meeting (2013)

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