title: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
authors.forename: Hejia Henry
authors.initials: HH
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA, 19104, USA.
authors.forename: Burcin
authors.initials: B
authors.lastname: Altun
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA, 19104, USA.
authors.forename: Kido
authors.initials: K
authors.lastname: Nwe
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA, 19104, USA.
authors.forename: Andrew
authors.identifier: 0000-0001-7758-1753
authors.initials: A
authors.lastname: Tsourkas
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; C000625572:SpyCatcher peptide; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: bioconjugation; expressed protein ligation; protein engineering; protein modifications; sortase