Overcoming the Limitations of Sortase with Proximity-Based Sortase-Mediated Ligation (PBSL)

  • Hejia Henry Wang
  • Andrew TsourkasEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2008)


S. aureus sortase A (SrtA), a calcium-dependent bacterial transpeptidase, is commonly used to site-specifically label proteins containing a LPXTG SrtA recognition motif with a wide array of chemical moieties. A major limitation of sortase-mediated labeling, however, is SrtA’s poor binding affinity to its recognition motif, resulting in long reaction times and poor ligation efficiencies. Here we describe proximity-based sortase-mediated ligation (PBSL), which utilizes the SpyTag-SpyCatcher peptide-protein pair to tether target proteins with a SrtA recognition motif to SrtA, dramatically increasing their local concentrations and overcoming this limitation.

Key words

Sortase SrtA SpyTag SpyCatcher Bioconjugation Proximity ligation Site-specific 


  1. 1.
    Krall N, da Cruz FP, Boutureira O, Bernardes GJL (2016) Site-selective protein-modification chemistry for basic biology and drug development. Nat Chem 8(2):102–112. CrossRefGoogle Scholar
  2. 2.
    Stephanopoulos N, Francis MB (2011) Choosing an effective protein bioconjugation strategy. Nat Chem Biol 7(12):876–884. CrossRefPubMedGoogle Scholar
  3. 3.
    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):2670–2671. CrossRefPubMedGoogle Scholar
  4. 4.
    Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL (2007) Sortagging: a versatile method for protein labeling. Nat Chem Biol 3(11):707–708. CrossRefPubMedGoogle Scholar
  5. 5.
    Haridas V, Sadanandan S, Dheepthi NU (2014) Sortase-based bio-organic strategies for macromolecular synthesis. Chembiochem 15(13):1857–1867. CrossRefPubMedGoogle Scholar
  6. 6.
    Ritzefeld M (2014) Sortagging: a robust and efficient chemoenzymatic ligation strategy. Chemistry 20(28):8516–8529. CrossRefPubMedGoogle Scholar
  7. 7.
    Jacobitz AW, Kattke MD, Wereszczynski J, Clubb RT (2017) Sortase transpeptidases: structural biology and catalytic mechanism. Adv Protein Chem Struct Biol 109:223–264. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Parthasarathy R, Subramanian S, Boder ET (2007) Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjug Chem 18(2):469–476. CrossRefPubMedGoogle Scholar
  9. 9.
    Chan L, Cross HF, She JK, Cavalli G, Martins HF, Neylon C (2007) Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation. PLoS One 2(11):e1164. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Heck T, Pham PH, Yerlikaya A, Thony-Meyer L, Richter M (2014) Sortase A catalyzed reaction pathways: a comparative study with six SrtA variants. Cat Sci Technol 4(9):2946–2956. CrossRefGoogle Scholar
  11. 11.
    Policarpo RL, Kang H, Liao XL, Rabideau AE, Simon MD, Pentelute BL (2014) Flow-based enzymatic ligation by sortase A. Angew Chem Int Edit 53(35):9203–9208. CrossRefGoogle Scholar
  12. 12.
    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):11188–11200. CrossRefPubMedGoogle Scholar
  13. 13.
    Warden-Rothman R, Caturegli I, Popik V, Tsourkas A (2013) Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step. Anal Chem 85(22):11090–11097. CrossRefPubMedGoogle Scholar
  14. 14.
    Wang HH, Altun B, Nwe K, Tsourkas A (2017) Proximity-based sortase-mediated ligation. Angew Chem Int Edit 56(19):5349–5352. CrossRefGoogle Scholar
  15. 15.
    Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci USA 109(12):E690–E697. CrossRefPubMedGoogle Scholar
  16. 16.
    Li L, Fierer JO, Rapoport TA, Howarth M (2014) Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag. J Mol Biol 426(2):309–317. CrossRefPubMedGoogle Scholar
  17. 17.
    Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41(1):207–234. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Graduate group in Biochemistry and Molecular BiophysicsUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of BioengineeringUniversity of PennsylvaniaPhiladelphiaUSA

Personalised recommendations