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

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Site-Specific Protein Labeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1266))

Abstract

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.

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Acknowledgements

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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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave, Rochester, NY, 14642, USA

    Maximilian Wei-Lin Popp

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  1. Maximilian Wei-Lin Popp
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Correspondence to Maximilian Wei-Lin Popp .

Editor information

Editors and Affiliations

  1. Department of Chemistry, École Normale Supérieure, Paris, France

    Arnaud Gautier

  2. Sensitive Farbstoffe GbR, Munich, Germany

    Marlon J. Hinner

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Popp, M.WL. (2015). Site-Specific Labeling of Proteins via Sortase: Protocols for the Molecular Biologist. In: Gautier, A., Hinner, M. (eds) Site-Specific Protein Labeling. Methods in Molecular Biology, vol 1266. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2272-7_13

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  • DOI: https://doi.org/10.1007/978-1-4939-2272-7_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2271-0

  • Online ISBN: 978-1-4939-2272-7

  • eBook Packages: Springer Protocols

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