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Proximity-Based Sortase-Mediated Ligation.

ABSTRACT: Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase?A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95?% by linking the target protein to SrtA using the SpyTag-SpyCatcher peptide-protein pair. By expressing the target protein with SpyTag C-terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher-SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.

SUBMITTER: Wang HH

PROVIDER: S-EPMC5537000 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Proximity-Based Sortase-Mediated Ligation.

Wang Hejia Henry HH Altun Burcin B Nwe Kido K Tsourkas Andrew A

Angewandte Chemie (International ed. in English) 20170404 19

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein ...[more]

PMID: 28374553

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Sortase-Mediated Ligation of Purely Artificial Building Blocks.

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Project description: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.

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Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation.

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