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Overcoming the Limitations of Sortase with Proximity-Based Sortase-Mediated Ligation (PBSL)

  • Hejia Henry Wang
  • Andrew TsourkasEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2008)

Abstract

S. aureus sortase A (SrtA), a calcium-dependent bacterial transpeptidase, is commonly used to site-specifically label proteins containing a LPXTG SrtA recognition motif with a wide array of chemical moieties. A major limitation of sortase-mediated labeling, however, is SrtA’s poor binding affinity to its recognition motif, resulting in long reaction times and poor ligation efficiencies. Here we describe proximity-based sortase-mediated ligation (PBSL), which utilizes the SpyTag-SpyCatcher peptide-protein pair to tether target proteins with a SrtA recognition motif to SrtA, dramatically increasing their local concentrations and overcoming this limitation.

Key words

Sortase SrtA SpyTag SpyCatcher Bioconjugation Proximity ligation Site-specific 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Graduate group in Biochemistry and Molecular BiophysicsUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of BioengineeringUniversity of PennsylvaniaPhiladelphiaUSA

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