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Proximity‐Based Sortase‐Mediated Ligation
Author(s) -
Wang Hejia Henry,
Altun Burcin,
Nwe Kido,
Tsourkas Andrew
Publication year - 2017
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201701419
Subject(s) - sortase a , bioconjugation , sortase , chemistry , ligation , peptide , fusion protein , biochemistry , combinatorial chemistry , covalent bond , native chemical ligation , target protein , bacterial protein , cysteine , microbiology and biotechnology , recombinant dna , biology , enzyme , organic chemistry , gene
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.