Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems

Autoři: Jakob C. Stüber aff001;  Andreas Plückthun aff001
Působiště autorů: Department of Biochemistry, University of Zurich, Winterthurerstrasse, Zurich, Switzerland aff001
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226579



Fluorescent labeling of specific cell-surface proteins enables a manifold of techniques to study their function in health and disease. A frequently cited family of methods employs phosphopantetheinyl transferases (PPTases) to attach probes, provided as conjugates of Coenzyme A. This method appears attractive, as only short peptide tags genetically fused to the protein of interest are needed as conjugation sites. Here, we describe observations we made when evaluating such protocols for delicate single-molecule applications where we require a particular combination of dyes, low background binding or low labeling of other proteins, and a high degree of labeling.


When we tested a PPTase-acceptor peptide couple with several experimental protocols and various CoA conjugates for labeling of a protein on the cell surface, we noticed substantial non-specific labeling. For the first time, we provide here a quantification of the non-specific fraction of the signals obtained using appropriate controls. We further present evidence that this background is due to CoA-dye conjugates entering the cell, where they may be covalently attached to endogenous proteins. However, when studying cell-surface proteins, most fluorescent readouts require that labeling is strictly limited to the protein of interest located at the cell surface. While such data have so far been missing in the literature, they suggest that for applications where labeling of unwanted molecules would affect the conclusions, researchers need to be aware of this potential non-specificity of PPTase methods when selecting a labeling strategy. We show, again by quantitative comparison, that the HaloTag is a viable alternative.

Klíčová slova:

Cell fusion – Cell staining – Enzymes – Flow cytometry – Genetic engineering – Signal peptides – HEK 293 cells – Fluorescent dyes


1. Jensen EC. Use of Fluorescent Probes: Their Effect on Cell Biology and Limitations. Anat Rec. 2012;295:2031–6.

2. Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005;2:905–9. doi: 10.1038/nmeth819 16299475

3. Yano Y, Matsuzaki K. Tag-probe labeling methods for live-cell imaging of membrane proteins. Biochim Biophys Acta. 2009;1788:2124–31. doi: 10.1016/j.bbamem.2009.07.017 19646952

4. Beld J, Sonnenschein EC, Vickery CR, Noel JP, Burkart MD. The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life. Nat Prod Rep. 2014;31:61–108. doi: 10.1039/c3np70054b 24292120

5. George N, Pick H, Vogel H, Johnsson N, Johnsson K. Specific Labeling of Cell Surface Proteins with Chemically Diverse Compounds. J Am Chem Soc. 2004;126:8896–7. doi: 10.1021/ja048396s 15264811

6. Yin J, Liu F, Li X, Walsh CT. Labeling proteins with small molecules by site-specific posttranslational modification. J Am Chem Soc. 2004;126:7754–5. doi: 10.1021/ja047749k 15212504

7. Zhou Z, Cironi P, Lin AJ, Xu YQ, Hrvatin S, Golan DE, et al. Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem Biol. 2007;2:337–46. doi: 10.1021/cb700054k 17465518

8. Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, et al. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem Biol. 2008;3:373–82. doi: 10.1021/cb800025k 18533659

9. Yin J, Lin AJ, Golan DE, Walsh CT. Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat Protoc. 2006;1:280–5. doi: 10.1038/nprot.2006.43 17406245

10. Yin J, Straight PD, McLoughlin SM, Zhou Z, Lin AJ, Golan DE, et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc Natl Acad Sci U S A. 2005;102:15815–20. doi: 10.1073/pnas.0507705102 16236721

11. Ioannidis JPA. Why Most Published Research Findings Are False. PLoS Med. 2005;2:e124. doi: 10.1371/journal.pmed.0020124 16060722

12. Ioannidis JPA. How to Make More Published Research True. PLoS Med. 2014;11:e1001747. doi: 10.1371/journal.pmed.1001747 25334033

13. Prinz F, Schlange T, Asadullah K. Believe it or not: how much can we rely on published data on potential drug targets? Nature Reviews Drug Discovery. 2011;10:712. doi: 10.1038/nrd3439-c1 21892149

14. Scraping cell cultures instead of using trypsin. 2013 [Cited 21 Feb 2018]. In: LGC Standards-ATCC [Internet]. Available from:

15. McNeil PL, Murphy RF, Lanni F, Taylor DL. A method for incorporating macromolecules into adherent cells. J Cell Biol. 1984;98:1556–64. doi: 10.1083/jcb.98.4.1556 6201494

16. Lukinavičius G, Umezawa K, Olivier N, Honigmann A, Yang G, Plass T, et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem. 2013;5:132–9. doi: 10.1038/nchem.1546 23344448

17. Humpert ML, Tzouros M, Thelen S, Bignon A, Levoye A, Arenzana-Seisdedos F, et al. Complementary methods provide evidence for the expression of CXCR7 on human B cells. Proteomics. 2012;12:1938–48. doi: 10.1002/pmic.201100581 22623068

18. Yao JZ, Uttamapinant C, Poloukhtine A, Baskin JM, Codelli JA, Sletten EM, et al. Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition. J Am Chem Soc. 2012;134:3720–8. doi: 10.1021/ja208090p 22239252

19. Zanetti-Domingues LC, Tynan CJ, Rolfe DJ, Clarke DT, Martin-Fernandez M. Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding. PLOS ONE. 2013;8:e74200. doi: 10.1371/journal.pone.0074200 24066121

20. Marchetti L, De Nadai T, Bonsignore F, Calvello M, Signore G, Viegi A, et al. Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags. PLOS ONE. 2014;9:18.

21. Hinner MJ, Johnsson K. How to obtain labeled proteins and what to do with them. Curr Opin Biotechnol. 2010;21:766–76. doi: 10.1016/j.copbio.2010.09.011 21030243

22. Parris KD, Lin L, Tam A, Mathew R, Hixon J, Stahl M, et al. Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure. 2000;8:883–95. doi: 10.1016/s0969-2126(00)00178-7 10997907

23. Mofid MR, Finking R, Essen LO, Marahiel MA. Structure-Based Mutational Analysis of the 4‘-Phosphopantetheinyl Transferases Sfp from Bacillus subtilis: Carrier Protein Recognition and Reaction Mechanism. Biochemistry. 2004;43:4128–36. doi: 10.1021/bi036013h 15065855

24. Splittgerber AG. Simplified treatment of two-substrate enzyme kinetics. J Chem Educ. 1983;60:651.

25. Gehring AM, Lambalot RH, Vogel KW, Drueckhammer DG, Walsh CT. 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. 1997;4:17–24. doi: 10.1016/s1074-5521(97)90233-7 9070424

26. ACP-Surface Starter Kit. 2017 [Cited 28 Feb 2018]. In: New England Biolabs [Internet]. Available from:

27. Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL. Sortagging: a versatile method for protein labeling. Nat Chem Biol. 2007;3:707–8. doi: 10.1038/nchembio.2007.31 17891153

28. Reddington SC, Howarth M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol. 2015;29:94–9. doi: 10.1016/j.cbpa.2015.10.002 26517567

29. Lin CW, Ting AY. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J Am Chem Soc. 2006;128:4542–3. doi: 10.1021/ja0604111 16594669

30. Sun X, Zhang A, Baker B, Sun L, Howard A, Buswell J, et al. Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging. ChemBioChem. 2011;12:2217–26. doi: 10.1002/cbic.201100173 21793150

31. Grimm JB, Brown TA, English BP, Lionnet T, Lavis LD. Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments. In: Erfle H, editor. Super-Resolution Microscopy: Methods and Protocols. New York: Springer; 2017. pp. 179–88.

32. Kosaka N, Ogawa M, Choyke PL, Karassina N, Corona C, McDougall M, et al. In Vivo Stable Tumor-Specific Painting in Various Colors Using Dehalogenase-Based Protein-Tag Fluorescent Ligands. Bioconj Chem. 2009;20:1367–74.

33. Stüber JC, Kast F, Plückthun A. High-Throughput Quantification of Surface Protein Internalization and Degradation. ACS Chem Biol. 2019;14:1154–63. doi: 10.1021/acschembio.9b00016 31050891

34. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006;24:79–88. doi: 10.1038/nbt1172 16369541

35. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. doi: 10.1038/nmeth.2019 22743772

Článek vyšel v časopise


2019 Číslo 12