Site-Specific Labeling of Proteins via Sortase: Protocols for the Molecular Biologist

  • Maximilian Wei-Lin PoppEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1266)


Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5o) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5o variant.

Key words

Sortase Transpeptidation Site-specific labeling Sortagging Chemoenzymatic labeling VHH 



MWP is supported by an HHMI fellowship from the Damon Runyon Cancer Research Foundation, DRG-2119-12. I gratefully thank Hidde Ploegh, David Liu, and Lynne Maquat for advice, reagents, and support.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry and Biophysics, School of Medicine and DentistryUniversity of RochesterRochesterUSA

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