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Site-specific N-terminal labeling of proteins using sortase-mediated reactions

Nature Protocols volume 8, pages 1800–1807 (2013)Cite this article

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Abstract

This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Staphylococcus aureus sortase A) or LPXTA (for Streptococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.

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Figure 1: N-terminal labeling of proteins.
Figure 2: Specific labeling of cholera toxin B subunit (CTxB) at its N terminus with a fluorophore using S. aureus sortase A.

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Change history

  • 06 December 2013

     In the version of this article initially published, the genus names for the bacteria were transposed in the abstract so that they incorrectly read Streptococcus aureus and Staphylococcus pyogenes instead of Staphylococcus aureus and Streptococcus pyogenes. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by funding from The Netherlands Organisation for Scientific Research (to M.D.W.) and the US National Institutes of Health (NIH; grant no. RO1 AI087879 to H.L.P.).

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Author notes

  1. Martin D Witte

    Present address: Present address: Bio-Organic Chemistry, Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands.,

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Christopher S Theile, Martin D Witte, Annet E M Blom, Lenka Kundrat, Hidde L Ploegh & Carla P Guimaraes

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Hidde L Ploegh

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Contributions

C.S.T., C.P.G. and H.L.P. conceived of and drafted the manuscript. C.S.T., M.D.W., A.E.M.B., L.K. and C.P.G. participated in the optimization of the protocols and wrote the manuscript. H.L.P. revised the manuscript.

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Correspondence to Hidde L Ploegh or Carla P Guimaraes.

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Theile, C., Witte, M., Blom, A. et al. Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat Protoc 8, 1800–1807 (2013). https://doi.org/10.1038/nprot.2013.102

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