Abstract
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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Acknowledgements
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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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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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): https://doi.org/10.1039/D0GC01366H
Du, J. et al. Nat. Commun. 11, 1575 (2020): https://doi.org/10.1038/s41467-020-15418-2
Rehm, F. B. H. et al. Angew. Chem. Int. Ed.(2020): https://doi.org/10.1002/anie.202013584
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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). https://doi.org/10.1038/s41596-020-00483-0
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DOI: https://doi.org/10.1038/s41596-020-00483-0
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