Skip to main content
SpringerLink
Log in

Enzymatic Ligation of Disulfide-Rich Animal Venom Peptides: Using Sortase A to Form Double-Knotted Peptides

  • Protocol
  • First Online:
Peptide Conjugation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2355))

  • 1276 Accesses

Abstract

Sortase A is a thiol transpeptidase expressed by Gram-positive bacteria. This enzyme is capable of site-specifically ligating peptides containing the C-terminal recognition motif LPXTG to peptides containing an N-terminal polyglycine sequence, forming a native peptide bond. Here, we describe the preparation and application of sortase A to the ligation of two individually folded disulfide-rich animal venom peptides in order to form a heterodimeric double-knotted peptide with a native peptide linker. This method is mild enough to preserve the structures and disulfide connectivities of the peptides during ligation. We employed a highly efficient sortase A pentamutant (SrtA5°), which brings the reaction to completion within 15 min with a ~50–80% yield of ligated peptide.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Schneewind O, Model P, Fischetti VA (1992) Sorting of protein A to the staphylococcal cell wall. Cell 70(2):267–281

    Article  CAS  Google Scholar 

  2. Ojeda PG, Chan LY, Poth AG, Wang CK, Craik DJ (2014) The role of disulfide bonds in structure and activity of chlorotoxin. Future Med Chem 6(15):1617–1628

    Article  CAS  Google Scholar 

  3. Herzig V, King GF (2015) The cystine knot is responsible for the exceptional stability of the insecticidal spider toxin omega-hexatoxin-Hv1a. Toxins (Basel) 7(10):4366–4380. https://doi.org/10.3390/toxins7104366

    Article  CAS  Google Scholar 

  4. Kikuchi K, Sugiura M, Kimura T (2015) High proteolytic resistance of spider-derived inhibitor cystine knots. Int J Pept 2015:537508. https://doi.org/10.1155/2015/537508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Agwa AJ, Huang YH, Craik DJ, Henriques ST, Schroeder CI (2017) Lengths of the C-terminus and interconnecting loops impact stability of spider-derived gating modifier toxins. Toxins 9(8):248–262. https://doi.org/10.3390/toxins9080248

    Article  CAS  PubMed Central  Google Scholar 

  6. Bohlen CJ, Priel A, Zhou S, King D, Siemens J, Julius D (2010) A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain. Cell 141(5):834–845. https://doi.org/10.1016/j.cell.2010.03.052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chassagnon IR, McCarthy CA, Chin YK, Pineda SS, Keramidas A, Mobli M, Pham V, De Silva TM, Lynch JW, Widdop RE, Rash LD, King GF (2017) Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a. Proc Natl Acad Sci U S A 114(14):3750–3755. https://doi.org/10.1073/pnas.1614728114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Vassilevski AA, Fedorova IM, Maleeva EE, Korolkova YV, Efimova SS, Samsonova OV, Schagina LV, Feofanov AV, Magazanik LG, Grishin EV (2010) Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide. J Biol Chem 285(42):32293–32302. https://doi.org/10.1074/jbc.M110.104265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Maxwell M, Undheim EAB, Mobli M (2018) Secreted cysteine-rich repeat proteins “SCREPs”: a novel multi-domain architecture. Front Pharmacol 9:1333. https://doi.org/10.3389/fphar.2018.01333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Murray JK, Biswas K, Holder JR, Zou A, Ligutti J, Liu D, Poppe L, Andrews KL, Lin FF, Meng SY, Moyer BD, McDonough SI, Miranda LP (2015) Sustained inhibition of the NaV1.7 sodium channel by engineered dimers of the domain II binding peptide GpTx-1. Bioorg Med Chem Lett 25(21):4866–4871. https://doi.org/10.1016/j.bmcl.2015.06.033

    Article  CAS  PubMed  Google Scholar 

  11. Agwa AJ, Blomster LV, Craik DJ, King GF, Schroeder CI (2018) Efficient enzymatic ligation of inhibitor cystine knot spider venom peptides: using sortase A to form double-knottins that probe voltage-gated sodium channel NaV1.7. Bioconjug Chem 29(10):3309–3319. https://doi.org/10.1021/acs.bioconjchem.8b00505

    Article  CAS  PubMed  Google Scholar 

  12. Tran HNT, Tran P, Deuis JR, Agwa AJ, Zhang AH, Vetter I, Schroeder CI (2020) Enzymatic ligation of a pore blocker toxin and a gating modifier toxin: creating double-knotted peptides with improved sodium channel NaV1.7 inhibition. Bioconjug Chem 31(1):64–73. https://doi.org/10.1021/acs.bioconjchem.9b00744

    Article  CAS  PubMed  Google Scholar 

  13. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779

    Article  CAS  Google Scholar 

  14. Cistrone PA, Bird MJ, Flood DT, Silvestri AP, Hintzen JCJ, Thompson DA, Dawson PE (2019) Native chemical ligation of peptides and proteins. Curr Protoc Chem Biol 11(1): e6. https://doi.org/10.1002/cpch.61

  15. 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):11399–11404. https://doi.org/10.1073/pnas.1101046108

    Article  PubMed  PubMed Central  Google Scholar 

  16. Agwa AJ, Craik DJ, Schroeder CI (2019) Cyclizing disulfide-rich peptides using sortase a. In: Nuijens T, Schmidt M (eds) Enzyme-mediated ligation methods, Methods in molecular biology, vol 2012. Humana, New York, NY, pp 29–41

    Chapter  Google Scholar 

  17. Popp MW, Antos JM, Ploegh HL (2009) Site-specific protein labeling via sortase-mediated transpeptidation. Curr Protoc Protein Sci Chapter 15:Unit 15.13. https://doi.org/10.1002/0471140864.ps1503s56

    Article  Google Scholar 

  18. 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

    Article  CAS  Google Scholar 

  19. Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182(2):319–326

    Article  CAS  Google Scholar 

  20. Williamson DJ, Fascione MA, Webb ME, Turnbull WB (2012) Efficient N-terminal labeling of proteins by use of sortase. Angew Chem Int Ed Engl 51(37):9377–9380. https://doi.org/10.1002/anie.201204538

    Article  CAS  PubMed  Google Scholar 

  21. Peschel A, Cardoso FC, Walker AA, Durek T, Stone MRL, Emidio NB, Dawson PE, Muttenthaler M, King GF (2020) Two for the price of one: Heterobivalent ligand design targeting two binding sites on voltage-gated sodium channels slows ligand dissociation and enhances potency. J Med Chem 63(21):12773–12785. https://doi.org/10.1021/acs.jmedchem.0c01107

Download references

Acknowledgments

This work was supported by the Australian National Health and Medical Research Council (NHMRC) through a Project Grant (APP1080405), and an Australian Research Council (ARC) Future Fellowship (FT160100055) to C.I.S. P.T is supported by a University of Queensland Research Training Scholarship. We thank Prof. David Liu at Howard Hughes Medical School at Harvard University for providing the SrtA5° plasmid, Mr. Alan Zhang at the University of Queensland Centre for Advanced Imaging for assistance with SrtA5° expression, and Ms. Hue N.T. Tran for assistance with peptide synthesis.

Author information

Authors and Affiliations

  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia

    Poanna Tran & Christina I. Schroeder

  2. National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Christina I. Schroeder

Authors
  1. Poanna Tran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christina I. Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christina I. Schroeder .

Editor information

Editors and Affiliations

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, Australia

    Waleed M. Hussein

  2. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, Australia

    Rachel J. Stephenson

  3. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, Australia

    Istvan Toth

Rights and permissions

Reprints and permissions

Copyright information

© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tran, P., Schroeder, C.I. (2021). Enzymatic Ligation of Disulfide-Rich Animal Venom Peptides: Using Sortase A to Form Double-Knotted Peptides. In: Hussein, W.M., Stephenson, R.J., Toth, I. (eds) Peptide Conjugation. Methods in Molecular Biology, vol 2355. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1617-8_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1617-8_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1616-1

  • Online ISBN: 978-1-0716-1617-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation