End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation.
End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation.
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通过分选酶介导的连接对重组血栓调节蛋白进行终点固定。
DOI：
10.1021/bc200661w
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发表时间：
2012-03-21
影响因子：
4.7
通讯作者：
Sun, Xue-Long
中科院分区：
文献类型：
--
作者：
Jiang, Rui;Weingart, Jacob;Zhang, Hailong;Ma, Yong;Sun, Xue-Long
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 N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 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 is rapid occurring 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.
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影响因子：
3.6
作者：
Han, HS;Yang, SL;Shi, GY
通讯作者：
Shi, GY
影响因子：
2.9
作者：
Kruger, RG;Otvos, B;McCafferty, DG
通讯作者：
McCafferty, DG
影响因子：
2.9
作者：
Lu, Changsheng;Zhu, Jie;Zhang, Zhiwen
通讯作者：
Zhang, Zhiwen
影响因子：
14
作者：
Vasilets, VN;Hermel, G;Jacobasch, HJ
通讯作者：
Jacobasch, HJ
DOI：
10.1073/pnas.96.22.12424
发表时间：
1999-10-26
影响因子：
11.1
作者：
Ton-That, H;Liu, G;Schneewind, O
通讯作者：
Schneewind, O