Skip to main content
SpringerLink
Log in

Cyclizing Disulfide-Rich Peptides Using Sortase A

  • Protocol
  • First Online:
Enzyme-Mediated Ligation Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2012))

Abstract

Sortase A (SrtA) is an enzyme obtained from Staphylococcus aureus that catalyzes site-specific transpeptidation of surface proteins to the bacterial cell membrane. SrtA recognizes an LPXTG amino acid motif and cleaves between the Thr and Gly to form a thioester-linked acyl–enzyme intermediate. The intermediate is resolved in the presence of a nucleophilic N-terminal polyglycine resulting in ligation of the acyl donor to the polyglycine acceptor. Here we describe the application of SrtA as a tool for the cyclization of disulfide-rich peptides. Reactions are typically tailored to each disulfide-rich peptide with optimal conditions producing yields of 40–50% cyclized 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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
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. Craik DJ, Du J (2017) Cyclotides as drug design scaffolds. Curr Opin Chem Biol 38:8–16. https://doi.org/10.1016/j.cbpa.2017.01.018

    Article  CAS  Google Scholar 

  2. Wang CK, Craik DJ (2018) Designing macrocyclic disulfide-rich peptides for biotechnological applications. Nat Chem Biol 14:417–427. https://doi.org/10.1038/s41589-018-0039-y

    Article  CAS  Google Scholar 

  3. Innovate-AG Sero-X: the active. Innovate AG. http://innovate-ag.com.au/sero-x/the-active/. Accessed 14 May 2018

  4. Cheneval O, Schroeder CI, Durek T, Walsh P, Huang Y-H, Liras S, Price DA, Craik DJ (2014) Fmoc-based synthesis of disulfide-rich cyclic peptides. J Org Chem 79:5538–5544. https://doi.org/10.1021/jo500699m

    Article  CAS  Google Scholar 

  5. Hackeng TM, Griffin JH, Dawson PE (1999) Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc Natl Acad Sci U S A 96:10068–10073. https://doi.org/10.1073/pnas.96.18.10068

    Article  CAS  Google Scholar 

  6. Mende F, Seitz O (2011) 9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide alpha-thioesters. Angew Chem 50:1232–1240. https://doi.org/10.1002/anie.201005180

    Article  CAS  Google Scholar 

  7. Akcan M, Stroud MR, Hansen SJ, Clark RJ, Daly NL, Craik DJ, Olson JM (2011) Chemical re-engineering of chlorotoxin improves bioconjugation properties for tumor imaging and targeted therapy. J Med Chem 54:782–787. https://doi.org/10.1021/jm101018r

    Article  CAS  Google Scholar 

  8. Mao H, Hart SA, Schink A, Pollok BA (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 126:2670–2671. https://doi.org/10.1021/ja039915e

    Article  CAS  Google Scholar 

  9. Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AEM, Ploegh HL (2013) Site-specific C-terminal internal loop labeling of proteins using sortase-mediated reactions. Nat Protoc 8:1787–1799. https://doi.org/10.1038/nprot.2013.101

    Article  CAS  Google Scholar 

  10. Theile CS, Witte MD, Blom AEM, Kundrat L, Ploegh HL, Guimaraes CP (2013) Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat Protoc 8:1800–1807. https://doi.org/10.1038/nprot.2013.102

    Article  CAS  Google Scholar 

  11. Popp MW, Dougan SK, Chuang T-Y, Spooner E, Ploegh HL (2011) Sortase-catalyzed transformations that improve the properties of cytokines. Proc Natl Acad Sci U S A 108:3169–3174. https://doi.org/10.1073/pnas.1016863108

    Article  Google Scholar 

  12. Bolscher JG, Oudhoff MJ, Nazmi K, Antos JM, Guimaraes CP, Spooner E, Haney EF, Garcia Vallejo JJ, Vogel HJ, van’t Hof W, Ploegh HL, Veerman EC (2011) Sortase A as a tool for high-yield histatin cyclization. FASEB J 25:2650–2658. doi:https://doi.org/10.1096/fj.11-182212

    Article  CAS  Google Scholar 

  13. Jia X, Kwon S, Wang C-IA, Huang Y-H, Chan LY, Tan CC, Rosengren KJ, Mulvenna JP, Schroeder CI, Craik DJ (2014) Semienzymatic cyclization of disulfide-rich peptides using sortase A. J Biol Chem 289:6627–6638. https://doi.org/10.1074/jbc.M113.539262

    Article  CAS  Google Scholar 

  14. 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 108:11399–11404. https://doi.org/10.1073/pnas.1101046108

    Article  Google Scholar 

  15. 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. https://doi.org/10.1021/acs.bioconjchem.8b00505

    Article  CAS  Google Scholar 

  16. Ilangovan U, Ton-That H, Iwahara J, Schneewind O, Clubb RT (2001) Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus. Proc Natl Acad Sci U S A 98:6056–6061. https://doi.org/10.1073/pnas.101064198

    Article  CAS  Google Scholar 

  17. Kwon S, Bosmans F, Kaas Q, Cheneval O, Conibear AC, Rosengren KJ, Wang CK, Schroeder CI, Craik DJ (2016) Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide. Biotechnol Bioeng 113:2202–2212. https://doi.org/10.1002/bit.25993

    Article  CAS  Google Scholar 

  18. 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

  19. Witte MD, Wu T, Guimaraes CP, Theile CS, Blom AE, Ingram JR, Li Z, Kundrat L, Goldberg SD, Ploegh HL (2015) Site-specific protein modification using immobilized sortase in batch and continuous-flow systems. Nat Protoc 10:508–516. https://doi.org/10.1038/nprot.2015.026

    Article  CAS  Google Scholar 

  20. Steinhagen M, Zunker K, Nordsieck K, Beck-Sickinger AG (2013) Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus. Bioorg Med Chem 21:3504–3510. https://doi.org/10.1016/j.bmc.2013.03.039

    Article  CAS  Google Scholar 

  21. Kaiser E, Colescott RL, Bossinger CD, Cook PI (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34:595–598. https://doi.org/10.1016/0003-2697(70)90146-6

    Article  CAS  Google Scholar 

  22. Ghani HA, Henriques ST, Huang YH, Swedberg JE, Schroeder CI, Craik DJ (2017) Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies. Pept Sci 108:e22927. https://doi.org/10.1002/bip.22927

    Article  CAS  Google Scholar 

  23. Agwa AJ, Peigneur S, Chow CY, Lawrence N, Craik DJ, Tytgat J, King GF, Henriques ST, Schroeder CI (2018) Gating modifier toxins isolated from spider venom: modulation of voltage-gated sodium channels and the role of lipid membranes. J Biol Chem 293:9041–9052. https://doi.org/10.1074/jbc.RA118.002553

    Article  CAS  Google Scholar 

  24. Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326. https://doi.org/10.1016/0003-2697(89)90602-7

    Article  CAS  Google Scholar 

  25. Conibear AC, Daly NL, Craik DJ (2012) Quantification of small cyclic disulfide-rich peptides. Pept Sci 98:518–524. https://doi.org/10.1002/bip.22121

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by project grant (APP1047857) to D.J.C and C.I.S. from the Australian National Health and Medical Research Council (NMHRC). C.I.S is an Australian Research Council (ARC) Future Fellow (FT160100055), D.J.C is an ARC Australian Laureate Fellow (FL150100146), and A.J.A is supported by a University of Queensland International postgraduate student scholarship. Special thanks to Prof David Liu (Department of Chemistry and Chemical Biology, Harvard University), for SrtA5° expression plasmid and to Mr. Olivier Cheneval, Dr. Soohyun Kwon, Mr. Thomas Dash (Institute for Molecular Bioscience, University of Queensland), and Mr. Alan Zhang (Centre for Advanced Imaging, University of Queensland) for lab notes and scientific discussions.

Author information

Authors and Affiliations

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

    Akello J. Agwa, David J. Craik & Christina I. Schroeder

Authors
  1. Akello J. Agwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David J. Craik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christina I. Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to David J. Craik or Christina I. Schroeder .

Editor information

Editors and Affiliations

  1. EnzyPep B.V., Geleen, The Netherlands

    Timo Nuijens  & Marcel Schmidt  & 

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Agwa, A.J., Craik, D.J., Schroeder, C.I. (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. https://doi.org/10.1007/978-1-4939-9546-2_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9546-2_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9545-5

  • Online ISBN: 978-1-4939-9546-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation