Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

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

Subjects

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Guimaraes, C.P. et al. Identification of host cell factors required for intoxication through use of modified cholera toxin. J. Cell Biol. 195, 751–764 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Stephanopoulos, N. & Francis, M.B. Choosing an effective protein bioconjugation strategy. Nat. Chem. Biol. 7, 876–884 (2011).

    Article  CAS  PubMed  Google Scholar 

  4. Dirksen, A. & Dawson, P.E. Expanding the scope of chemoselective peptide ligations in chemical biology. Curr. Opin. Chem. Biol. 12, 760–766 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Dmochewitz, L. et al. A recombinant fusion toxin based on enzymatic inactive c3bot1 selectively targets macrophages. PLoS ONE 8, e54517 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jang, J.I. et al. Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice. Microbiol. Immunol. 56, 595–604 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Gellerman, G., Baskin, S., Galia, L., Gilad, Y. & Firer, M.A. Drug resistance to chlorambucil in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide. Anticancer Drugs 24, 112–119 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Baratova, L.A. et al. The topography of the surface of potato virus X: tritium planigraphy and immunological analysis. J. Gen. Virol. 73, 229–235 (1992).

    Article  CAS  PubMed  Google Scholar 

  9. Schwede, A., Jones, N., Engstler, M. & Carrington, M. The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes. Mol. Biochem. Parasit. 175, 201–204 (2011).

    Article  CAS  Google Scholar 

  10. Hess, G.T. et al. M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins. Bioconjugate Chem. 23, 1478–1487 (2012).

    Article  CAS  Google Scholar 

  11. Reyes-Turcu, F.E. et al. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197–1208 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Love, K.R., Pandya, R.K., Spooner, E. & Ploegh, H.L. Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery. ACS Chem. Biol. 4, 275–287 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Greenhalf, W., Stephan, C. & Chaudhuri, B. Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax-induced growth arrest and mortality in Saccharomyces cerevisiae. FEBS Lett. 380, 169–175 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Kataoka, T. et al. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension. J. Biol. Chem. 276, 19548–19554 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Miyadera, T. & Kosower, E.M. Receptor site labeling through functional groups. 2. Reactivity of maleimide groups. J. Med. Chem. 15, 534–537 (1972).

    Article  CAS  PubMed  Google Scholar 

  16. Palmer, M., Buchkremer, M., Valeva, A. & Bhakdi, S. Cysteine-specific radioiodination of proteins with fluorescein maleimide. Anal. Biochem. 253, 175–179 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Hnatowich, D.J. et al. Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine. J. Nucl. Med. 39, 56–64 (1998).

    CAS  PubMed  Google Scholar 

  18. Rabuka, D., Rush, J.S., deHart, G.W., Wu, P. & Bertozzi, C.R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat. Protoc. 7, 1052–1067 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu, P. et al. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proc. Natl. Acad. Sci. USA 106, 3000–3005 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Raschke, T.M., Kho, J. & Marqusee, S. Confirmation of the hierarchical folding of RNase H: a protein engineering study. Nat. Struct. Biol. 6, 825–831 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Rowe, S.M., Miller, S. & Sorscher, E.J. Cystic fibrosis. N. Engl. J. Med. 352, 1992–2001 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Carrico, Z.M. et al. N-Terminal labeling of filamentous phage to create cancer marker imaging agents. ACS Nano. 6, 6675–6680 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Guimaraes, C.P. et al. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1787–1799 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Paterson, G.K. & Mitchell, T.J. The biology of Gram-positive sortase enzymes. Trends Microbiol. 12, 89–95 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Ton-That, H., Liu, G., Mazmanian, S.K., Faull, K.F. & Schneewind, O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc. Natl. Acad. Sci. USA 96, 12424–12429 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mazmanian, S.K., Liu, G., Ton-That, H. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Spirig, T., Weiner, E.M. & Clubb, R.T. Sortase enzymes in Gram-positive bacteria. Mol. Microbiol. 82, 1044–1059 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Popp, M.W., Antos, J.M. & Ploegh, H.L. Site-specific protein labeling via sortase-mediated transpeptidation. Curr. Protoc. Protein Sci. 56, 15.3.1–15.3.9 (2009).

    Article  Google Scholar 

  29. Popp, M.W. & Ploegh, H.L. Making and breaking peptide bonds: protein engineering using sortase. Angew Chem. Int. Ed. Engl. 50, 5024–5032 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Ton-That, H. & Schneewind, O. Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction. J. Biol. Chem. 274, 24316–24320 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Race, P.R. et al. Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism. J. Biol. Chem. 284, 6924–6933 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tanaka, T., Yamamoto, T., Tsukiji, S. & Nagamune, T. Site-specific protein modification on living cells catalyzed by sortase. Chembiochem 9, 802–807 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Yamamoto, T. & Nagamune, T. Expansion of the sortase-mediated labeling method for site-specific N-terminal labeling of cell surface proteins on living cells. Chem. Commun. 9, 1022–1024 (2009).

    Article  CAS  Google Scholar 

  34. Tsukiji, S. & Nagamune, T. Sortase-mediated ligation: a gift from Gram-positive bacteria to protein engineering. Chembiochem 10, 787–798 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Williamson, D.J., Fascione, M.A., Webb, M.E. & Turnbull, W.B. Efficient N-terminal labeling of proteins by use of sortase. Angew Chem. Int. Ed. Engl. 51, 9377–9380 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. Cudic, P. & Stawikowski, M. Peptidomimetics: Fmoc solid-phase pseudopeptide synthesis. Methods Mol. Biol. 494, 223–246 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Witte, M.D. et al. Production of unnaturally linked chimeric proteins using a combination of sortase -catalyzed transpeptidation and click chemistry. Nat. Protoc. 8, 1808–1819 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Witte, M.D. et al. Preparation of unnatural N-to-N and C-to-C protein fusions. Proc. Natl. Acad. Sci. USA 109, 11993–11998 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Antos, J.M. et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Barrett, A.J., Rawlings, N.D. & Woessner, J.F. Handbook of Proteolytic Enzymes (Academic Press, 1998).

  41. Liu, D., Xu, R., Dutta, K. & Cowburn, D. N-terminal cysteinyl proteins can be prepared using thrombin cleavage. FEBS Lett. 582, 1163–1167 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gisin, B.F. The monitoring of reactions in solid-phase peptide synthesis with picric acid. Anal. Chim. Acta. 58, 248–249 (1972).

    Article  CAS  PubMed  Google Scholar 

  43. Vojkovsky, T. Detection of secondary amines on solid phase. Pept. Res. 8, 236–237 (1995).

    CAS  PubMed  Google Scholar 

  44. Schagger, H. Tricine-SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).

    Article  PubMed  CAS  Google Scholar 

  45. Hess, G.T., Guimaraes, C.P., Spooner, E., Ploegh, H.L. & Belcher, A.M. Orthogonal labeling of M13 minor capsid proteins with DNA to self-assemble end-to-end multiphage structures. ACS Synth Biol., doi:10.1021/sb400019s (2013).

Download references

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.).

Author information

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

Authors
  1. Christopher S Theile
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin D Witte
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Annet E M Blom
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lenka Kundrat
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hidde L Ploegh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carla P Guimaraes
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Hidde L Ploegh or Carla P Guimaraes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.102

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing