title: End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation.
abstract: 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 (TM(456)) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM(456) 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 TM(456) was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM(456) was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM(456) was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity.
authors.affiliation: Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Jiang
authors.forename: Jacob
authors.initials: J
authors.lastname: Weingart
authors.forename: Hailong
authors.initials: H
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Ma
authors.forename: Xue-Long
authors.initials: XL
authors.lastname: Sun
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D018180:Thrombomodulin; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases