End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation

We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4–6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS–PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivit...