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Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases


Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.

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Fig. 1: Overview of the procedure.
Fig. 2: IMAC purified CPs, activation of AEPs and mass spectrometry validation of precursors and mature cyclic peptides.
Fig. 3: Structural and functional validation of recombinant MCoTI-II.

Data availability

Raw data are available in the supporting information associated with the primary research articles.


  1. Colgrave, M. L. & Craik, D. J. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43, 5965–5975 (2004).

    Article  CAS  Google Scholar 

  2. Colgrave, M. L., Korsinczky, M. J., Clark, R. J., Foley, F. & Craik, D. J. Sunflower trypsin inhibitor-1, proteolytic studies on a trypsin inhibitor peptide and its analogs. Biopolymers 94, 665–672 (2010).

    Article  CAS  Google Scholar 

  3. Isidro-Llobet, A. et al. Sustainability challenges in peptide synthesis and purification: from R&D to production. J. Org. Chem. 84, 4615–4628 (2019).

    Article  CAS  Google Scholar 

  4. Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).

    Article  CAS  Google Scholar 

  5. Yap, K. et al. An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides. Green Chem. 22, 5002–5016 (2020).

    Article  CAS  Google Scholar 

  6. Cheneval, O. et al. Fmoc-based synthesis of disulfide-rich cyclic peptides. J. Org. Chem. 79, 5538–5544 (2014).

    Article  CAS  Google Scholar 

  7. El Hamdaoui, Y. et al. Periplasmic expression of 4/7 α-conotoxin TxIA analogues in E. coli favours ribbon isomer formation—suggestion of a binding mode at the α7 nAChR. Front. Pharmacol 10, 577 (2019).

    Article  CAS  Google Scholar 

  8. Gasser, B. et al. Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol. 8, 191–208 (2013).

    Article  CAS  Google Scholar 

  9. Pokoj, S. et al. Pichia pastoris is superior to E. coli for the production of recombinant allergenic non-specific lipid-transfer proteins. Protein Expr. Purif. 69, 68–75 (2010).

    Article  CAS  Google Scholar 

  10. Cregg, J. M., Vedvick, T. S. & Raschke, W. C. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11, 905–910 (1993).

    CAS  PubMed  Google Scholar 

  11. Cregg, J. M., Cereghino, J. L., Shi, J. & Higgins, D. R. Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23–52 (2000).

    Article  CAS  Google Scholar 

  12. Gassler, T. et al. The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2. Nat. Biotechnol. 38, 210–216 (2020).

    Article  CAS  Google Scholar 

  13. Poon, S. et al. Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. J. Exp. Bot. 69, 633–641 (2018).

    Article  CAS  Google Scholar 

  14. Jagadish, K. et al. Recombinant expression and phenotypic screening of a bioactive cyclotide against α‐synuclein‐induced cytotoxicity in baker’s yeast. Angew. Chem. Int. Ed 54, 8390–8394 (2015).

    Article  CAS  Google Scholar 

  15. Li, Y., Aboye, T., Breindel, L., Shekhtman, A. & Camarero, J. A. Efficient recombinant expression of SFTI‐1 in bacterial cells using intein‐mediated protein trans‐splicing. Pept. Sci. 106, 818–824 (2016).

    Article  CAS  Google Scholar 

  16. Toplak, A., Nuijens, T., Quaedflieg, P. J., Wu, B. & Janssen, D. B. Peptiligase, an enzyme for efficient chemoenzymatic peptide synthesis and cyclization in water. Adv. Synth. Catal. 358, 2140–2147 (2016).

    Article  CAS  Google Scholar 

  17. Antos, J. M. et al. A straight path to circular proteins. J. Biol. Chem. 284, 16028–16036 (2009).

    Article  CAS  Google Scholar 

  18. Du, J. et al. A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors. Nat. Commun. 11, 1575 (2020).

    Article  CAS  Google Scholar 

  19. Harris, K. S. et al. Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat. Commun. 6, 10199 (2015).

    Article  CAS  Google Scholar 

  20. Nguyen, G. K. et al. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 10, 732–738 (2014).

    Article  CAS  Google Scholar 

  21. Lin-Cereghino, G. P. et al. The effect of alpha-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene 519, 311–317 (2013).

    Article  CAS  Google Scholar 

  22. Cabral, K. M., Almeida, M. S., Valente, A. P., Almeida, F. C. & Kurtenbach, E. Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease. Protein Expr. Purif. 31, 115–122 (2003).

    Article  CAS  Google Scholar 

  23. Yang, R. et al. Engineering a catalytically efficient recombinant protein ligase. J. Am. Chem. Soc. 139, 5351–5358 (2017).

    Article  CAS  Google Scholar 

  24. Tolner, B., Smith, L., Begent, R. H. & Chester, K. A. Production of recombinant protein in Pichia pastoris by fermentation. Nat. Protoc. 1, 1006–1021 (2006).

    Article  CAS  Google Scholar 

  25. Armishaw, C. J., Dutton, J. L., Craik, D. J. & Alewood, P. F. Establishing regiocontrol of disulfide bond isomers of α‐conotoxin ImI via the synthesis of N‐to‐C cyclic analogs. Peptide Sci. 94, 307–313 (2010).

    Article  CAS  Google Scholar 

  26. Lovelace, E. S. et al. Stabilization of α-conotoxin AuIB: influences of disulfide connectivity and backbone cyclization. Antioxid. Redox Signal. 14, 87–95 (2011).

    Article  CAS  Google Scholar 

  27. Lovelace, E. S. et al. Cyclic MrIA: a stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter. J. Med. Chem. 49, 6561–6568 (2006).

    Article  CAS  Google Scholar 

  28. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  Google Scholar 

  29. Jia, X. Y. et al. Semienzymatic cyclization of disulfide-rich peptides using sortase A. J. Biol. Chem. 289, 6627–6638 (2014).

    Article  CAS  Google Scholar 

  30. Wu, Z., Guo, X. & Guo, Z. Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides. ChemComm 47, 9218–9220 (2011).

    CAS  Google Scholar 

  31. Schmidt, M. et al. Efficient enzymatic cyclization of disulfide‐rich peptides by using peptide ligases. ChemBioChem 20, 1524–1529 (2019).

    CAS  PubMed  Google Scholar 

  32. Schmidt, M. et al. Omniligase‐1: a powerful tool for peptide head‐to‐tail cyclization. Adv. Synth. Catal. 359, 2050–2055 (2017).

    Article  CAS  Google Scholar 

  33. Hemu, X., Qiu, Y., Nguyen, G. K. & Tam, J. P. Total synthesis of circular bacteriocins by butelase 1. J. Am. Chem. Soc. 138, 6968–6971 (2016).

    Article  CAS  Google Scholar 

  34. Nguyen, G. K., Hemu, X., Quek, J. P. & Tam, J. P. Butelase-mediated macrocyclization of d-amino-acid-containing peptides. Angew. Chem. Int. Ed. Engl. 55, 12802–12806 (2016).

    Article  CAS  Google Scholar 

  35. Johnson, B. H. & Hecht, M. H. Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Biotechnology 12, 1357–1360 (1994).

    CAS  PubMed  Google Scholar 

  36. Feliu, J., Cubarsi, R. & Villaverde, A. Optimized release of recombinant proteins by ultrasonication of E. coli cells. Biotechnol. Bioeng. 58, 536–540 (1998).

    Article  CAS  Google Scholar 

  37. Rehm, F. B. H. et al. Site-specific sequential protein labeling catalyzed by a single recombinant ligase. J. Am. Chem. Soc. 141, 17388–17393 (2019).

    Article  CAS  Google Scholar 

  38. Rehm, F. B. H., Tyler, T. J., Yap, K., Durek, T. & Craik, D. J. Improved asparaginyl ligase‐catalyzed transpeptidation via selective nucleophile quenching. Angew. Chem. Int. Ed. (2020).

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This work was supported by Australian Research Council (ARC) Discovery Project Grants (DP150100443 and DP200101299 (T.D. and D.J.C.)) and an ARC Australian Laureate Fellowship (FL150100146 (D.J.C.)). The project accessed the facilities of the ARC Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012). We gratefully acknowledge A. Jones for assistance with mass spectrometry, and O. Cheneval and L. Y. Chan for assistance with peptide synthesis.

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Authors and Affiliations



K.Y., L.H.L.L., T.D. and D.J.C. designed the research; K.Y., J.D., F.B.H.R., S.R.T., Y.Z., J.X. and S.J.d.V. performed the research; K.Y., J.D. and C.K.W. analyzed data; and all authors contributed to writing the manuscript.

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Correspondence to Thomas Durek or David J. Craik.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Yiming Li, Louis Luk, Novalia Pishesha, Anshan Shan and Zhengding Su for their contribution to the peer review of this work.

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Key references using this protocol

Yap, K. et al. Green Chem. 22, 5002 – 5016 (2020):

Du, J. et al. Nat. Commun. 11, 1575 (2020):

Rehm, F. B. H. et al. Angew. Chem. Int. Ed.(2020):

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Yap, K., Du, J., Rehm, F.B.H. et al. Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases. Nat Protoc 16, 1740–1760 (2021).

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