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 protein labeling by Sfp phosphopantetheinyl transferase

Nature Protocols volume 1, pages 280–285 (2006)Cite this article

Abstract

Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe–CoA conjugates, (iii) construction of target protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe–CoA conjugates as the substrates of Sfp, 3 d for the cloning of target protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged proteins. Labeling of the PCP- or the ybbR-tagged proteins in cell lysates or on cell surfaces should require only 15–30 min.

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: Synthesis of biotin-CoA (4) and Alexa Fluor 488–CoA (5) using CoA (1) and maleimide-linked biotin (2) and Alexa Fluor 488 (3), respectively.
Figure 2: Laser confocal images of TRVb cells transiently transfected with TfR1-PCP, labeled with CoA-conjugated Alexa Fluor 488 (green) in the presence of Sfp, and incubated with Tf-Alexa 568 (red) for 1 min (top panels) or 30 min (bottom panels).
Figure 3: FRET analysis of Tf–TfR1 interactions in TRVb cells transfected with TfR1-PCP.
Figure 4: FRET analysis of Tf–TfR1 interactions in TRVb cells transfected with TfR1-PCP.

Similar content being viewed by others

References

  1. Lambalot, R.H. et al. A new enzyme superfamily—the phosphopantetheinyl transferases. Chem. Biol. 3, 923–936 (1996).

    CAS  PubMed  Google Scholar 

  2. Walsh, C.T., Gehring, A.M., Weinreb, P.H., Quadri, L.E. & Flugel, R.S. Post-translational modification of polyketide and nonribosomal peptide synthases. Curr. Opin. Chem. Biol. 1, 309–315 (1997).

    CAS  PubMed  Google Scholar 

  3. Belshaw, P.J., Walsh, C.T. & Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284, 486–489 (1999).

    CAS  PubMed  Google Scholar 

  4. La Clair, J.J., Foley, T.L., Schegg, T.R., Regan, C.M. & Burkart, M.D. Manipulation of carrier proteins in antibiotic biosynthesis. Chem. Biol. 11, 195–201 (2004).

    CAS  PubMed  Google Scholar 

  5. Sieber, S.A., Walsh, C.T. & Marahiel, M.A. Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains. J. Am. Chem. Soc. 125, 10862–10866 (2003).

    CAS  PubMed  Google Scholar 

  6. Vitali, F., Zerbe, K. & Robinson, J.A. Production of vancomycin aglycone conjugated to a peptide carrier domain derived from a biosynthetic non-ribosomal peptide synthetase. Chem. Commun. (Camb) 21, 2718–2719 (2003).

    Google Scholar 

  7. Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., Jr. & Walsh, C.T. Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959–968 (2000).

    CAS  PubMed  Google Scholar 

  8. Gehring, A.M., Lambalot, R.H., Vogel, K.W., Drueckhammer, D.G. & Walsh, C.T. Ability of Streptomyces spp. acyl carrier proteins and coenzyme A analogs to serve as substrates in vitro for E. coli holo-ACP synthase. Chem. Biol. 4, 17–24 (1997).

    CAS  PubMed  Google Scholar 

  9. Mofid, M.R., Finking, R. & Marahiel, M.A. Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4′-phosphopantetheinyl transferases AcpS and Sfp. J. Biol. Chem. 277, 17023–17031 (2002).

    CAS  PubMed  Google Scholar 

  10. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    CAS  PubMed  Google Scholar 

  11. Yin, J., Liu, F., Li, X. & Walsh, C.T. Labeling proteins with small molecules by site-specific posttranslational modification. J. Am. Chem. Soc. 126, 7754–7755 (2004).

    CAS  PubMed  Google Scholar 

  12. Yin, J., Liu, F., Schinke, M., Daly, C. & Walsh, C.T. Phagemid encoded small molecules for high throughput screening of chemical libraries. J. Am. Chem. Soc. 126, 13570–13571 (2004).

    CAS  PubMed  Google Scholar 

  13. Vivero-Pol, L., George, N., Krumm, H., Johnsson, K. & Johnsson, N. Multicolor imaging of cell surface proteins. J. Am. Chem. Soc. 127, 12770–12771 (2005).

    CAS  PubMed  Google Scholar 

  14. Yin, J. et al. Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp catalyzed site specific protein labeling. Chem. Biol. 12, 999–1006 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl. Acad. Sci. USA 102, 15815–15820 (2005).

    CAS  PubMed  Google Scholar 

  16. Hermanson, G.T. Bioconjugate Techniques (Academic Press, San Diego, 1995), p. 148.

    Google Scholar 

  17. Stachelhaus, T. & Walsh, C.T. Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase. Biochemistry 39, 5775–5787 (2000).

    CAS  PubMed  Google Scholar 

  18. Horton, R.M. et al. Gene splicing by overlap extension. Methods Enzymol. 217, 270–279 (1993).

    CAS  PubMed  Google Scholar 

  19. McGraw, T.E., Greenfield, L. & Maxfield, F.R. Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor. J. Cell. Biol. 105, 207–214 (1987).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Marianne Wessling-Resnick and Peter D. Buckett of Harvard School of Public Health for technical assistance and helpful discussions.

Author information

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Jun Yin, Alison J Lin, David E Golan & Christopher T Walsh

Authors
  1. Jun Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alison J Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David E Golan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher T Walsh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christopher T Walsh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yin, J., Lin, A., Golan, D. et al. Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat Protoc 1, 280–285 (2006). https://doi.org/10.1038/nprot.2006.43

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.43

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