Advertisement

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

  • Maximilian Wei-Lin PoppEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Sortase Transpeptidation Site-specific labeling Sortagging Chemoenzymatic labeling VHH 

Notes

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.

References

  1. 1.
    Marraffini LA, Dedent AC, Schneewind O (2006) Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70(1):192–221PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci U S A 96(22):12424–12429PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Connolly KM, Smith BT, Pilpa R, Ilangovan U, Jung ME, Clubb RT (2003) Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site. J Biol Chem 278(36):34061–34065PubMedCrossRefGoogle Scholar
  4. 4.
    Frankel BA, Kruger RG, Robinson DE, Kelleher NL, McCafferty DG (2005) Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry 44(33):11188–11200PubMedCrossRefGoogle Scholar
  5. 5.
    Chen I, Dorr BM, Liu DR (2011) A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci U S A 108(28):11399–11404PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Popp MW, Antos JM, Ploegh HL (2009) Site-specific protein labeling via sortase-mediated transpeptidation. Curr Protoc Protein Sci Chapter 15, Unit 15.3Google Scholar
  7. 7.
    Antos JM, Miller GM, Grotenbreg GM, Ploegh HL (2008) Lipid modification of proteins through sortase-catalyzed transpeptidation. J Am Chem Soc 130(48):16338–16343PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Yamamoto T, Nagamune T (2009) Expansion of the sortase-mediated labeling method for site-specific N-terminal labeling of cell surface proteins on living cells. Chem Commun (Camb) 9:1022–1024CrossRefGoogle Scholar
  9. 9.
    Antos JM, Chew GL, Guimaraes CP, Yoder NC, Grotenbreg GM, Popp MW, Ploegh HL (2009) Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J Am Chem Soc 131(31):10800–10801PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Guimaraes CP, Carette JE, Varadarajan M, Antos J, Popp MW, Spooner E, Brummelkamp TR, Ploegh HL (2011) Identification of host cell factors required for intoxication through use of modified cholera toxin. J Cell Biol 195(5):751–764PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Popp MW, Artavanis-Tsakonas K, Ploegh HL (2009) Substrate filtering by the active site crossover loop in UCHL3 revealed by sortagging and gain-of-function mutations. J Biol Chem 284(6):3593–3602PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Popp MW, Karssemeijer RA, Ploegh HL (2012) Chemoenzymatic site-specific labeling of influenza glycoproteins as a tool to observe virus budding in real time. PLoS Pathog 8(3):e1002604PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Antos JM, Popp MW, Ernst R, Chew GL, Spooner E, Ploegh HL (2009) A straight path to circular proteins. J Biol Chem 284(23):16028–16036PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Claessen JH, Witte MD, Yoder NC, Zhu AY, Spooner E, Ploegh HL (2013) Catch-and-release probes applied to semi-intact cells reveal ubiquitin-specific protease expression in Chlamydia trachomatis infection. Chembiochem 14(3):343–352PubMedCrossRefGoogle Scholar
  15. 15.
    Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL (2007) Sortagging: a versatile method for protein labeling. Nat Chem Biol 3(11):707–708PubMedCrossRefGoogle Scholar
  16. 16.
    Popp MW, Dougan SK, Chuang TY, Spooner E, Ploegh HL (2011) Sortase-catalyzed transformations that improve the properties of cytokines. Proc Natl Acad Sci U S A 108(8):3169–3174PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Swee LK, Guimaraes CP, Sehrawat S, Spooner E, Barrasa MI, Ploegh HL (2013) Sortase-mediated modification of alphaDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes. Proc Natl Acad Sci U S A 110(4):1428–1433PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Witte MD, Cragnolini JJ, Dougan SK, Yoder NC, Popp MW, Ploegh HL (2012) Preparation of unnatural N-to-N and C-to-C protein fusions. Proc Natl Acad Sci U S A 109(30):11993–11998PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Tanaka T, Yamamoto T, Tsukiji S, Nagamune T (2008) Site-specific protein modification on living cells catalyzed by Sortase. Chembiochem 9(5):802–807PubMedCrossRefGoogle Scholar
  20. 20.
    Mao H, Hart SA, Schink A, Pollok BA (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 126(9):2670–2671PubMedCrossRefGoogle Scholar
  21. 21.
    Hendricks GL, Weirich KL, Viswanathan K, Li J, Shriver ZH, Ashour J, Ploegh HL, Kurt-Jones EA, Fygenson DK, Finberg RW, Comolli JC, Wang JP (2013) Sialylneolacto-N-tetraose c (LSTc)-bearing liposomal decoys capture influenza A virus. J Biol Chem 288(12):8061–8073PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Tafesse FG, Sanyal S, Ashour J, Guimaraes CP, Hermansson M, Somerharju P, Ploegh HL (2013) Intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins. Proc Natl Acad Sci U S A 110(16):6406–6411PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Hess GT, Cragnolini JJ, Popp MW, Allen MA, Dougan SK, Spooner E, Ploegh HL, Belcher AM, Guimaraes CP (2012) M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins. Bioconjug Chem 23(7):1478–1487PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Esteban A, Popp MW, Vyas VK, Strijbis K, Ploegh HL, Fink GR (2011) Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages. Proc Natl Acad Sci U S A 108(34):14270–14275PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Hirota N, Yasuda D, Hashidate T, Yamamoto T, Yamaguchi S, Nagamune T, Nagase T, Shimizu T, Nakamura M (2010) Amino acid residues critical for endoplasmic reticulum export and trafficking of platelet-activating factor receptor. J Biol Chem 285(8):5931–5940PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Wu Z, Guo X, Wang Q, Swarts BM, Guo Z (2010) Sortase A-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymatic synthesis of GPI-anchored proteins. J Am Chem Soc 132(5):1567–1571PubMedCrossRefGoogle Scholar
  27. 27.
    Popp MW, Ploegh HL (2011) Making and breaking peptide bonds: protein engineering using sortase. Angew Chem Int Ed Engl 50(22):5024–5032PubMedCrossRefGoogle Scholar
  28. 28.
    Strijbis K, Spooner E, Ploegh HL (2012) Protein ligation in living cells using sortase. Traffic 13(6):780–789PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Hendrickx AP, Budzik JM, Oh SY, Schneewind O (2011) Architects at the bacterial surface—sortases and the assembly of pili with isopeptide bonds. Nat Rev Microbiol 9(3):166–176PubMedCrossRefGoogle Scholar
  30. 30.
    Ling JJ, Policarpo RL, Rabideau AE, Liao X, Pentelute BL (2012) Protein thioester synthesis enabled by sortase. J Am Chem Soc 134(26):10749–10752PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Pierce NW, Lee JE, Liu X, Sweredoski MJ, Graham RL, Larimore EA, Rome M, Zheng N, Clurman BE, Hess S, Shan SO, Deshaies RJ (2013) Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153(1):206–215PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Pos W, Sethi DK, Call MJ, Schulze MS, Anders AK, Pyrdol J, Wucherpfennig KW (2012) Crystal structure of the HLA-DM-HLA-DR1 complex defines mechanisms for rapid peptide selection. Cell 151(7):1557–1568PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Coin I, Beyermann M, Bienert M (2007) Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat Protoc 2(12):3247–3256PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry and Biophysics, School of Medicine and DentistryUniversity of RochesterRochesterUSA

Personalised recommendations