Abstract
The ability to precisely modify proteins and peptides is fundamental to studying their function and creating new variants or topologies with improved properties. Recent studies have transformed the scope of transpeptidases as versatile tools for site-specific modification of proteins and peptides. The engineered asparaginyl ligase OaAEP1 is an ultrafast transpeptidase that stands out owing to its ability to efficiently catalyze a diverse range of modifications that extend well beyond its natural function to generate backbone cyclic peptides in plants. In this Protocol Extension, we describe a framework for the design and application of noncanonical reactions catalyzed by OaAEP1 that provide access to engineered products with customized terminal or side-chain modifications. The reactions proceed cleanly under mild, nondenaturing conditions and can be applied to a broad array of substrates produced by chemical synthesis or recombinant expression, including folded proteins and peptides. After preparing the required substrates and reagents (~5 d) and expressing the recombinant enzyme in Escherichia coli (~3 d), OaAEP1-catalyzed reactions can be carried out in a matter of minutes to hours. We describe methods for installing non-native C-terminal modifications, including by conjugating commercially available nonpeptidic amines (reactive handles, carbohydrates and so on) or ligating a reversed (retro) substrate mimetic that enables production of genetically inaccessible C-to-C fusions. We also describe procedures for OaAEP1-catalyzed side-chain modification of proteins and peptides, which can be applied to generate side-chain-to-tail macrocyclic products, to label a specific side-chain amine with a dye or other reporter tag, or to produce defined protein–cyclic peptide fusions.
Key points
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This Protocol Extension describes a series of procedures for introducing different types of modifications into proteins or peptides using the asparaginyl ligase OaAEP1.
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The reactions are highly versatile and enable late-stage modification of folded proteins and peptides, providing direct access to products with diverse C-terminal or side-chain modifications that are currently challenging to achieve with other enzymes or chemical approaches.
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Data availability
Data supporting the individual procedures detailed in this protocol are included in the current article and in the published articles that form the basis for the protocol.
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Acknowledgements
Work in our laboratory on asparaginyl ligases is supported by a grant from the Australian Research Council (ARC, DP200101299) and by access to the facilities of the ARC Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012). D.J.C. and F.B.H.R. are supported by National Health & Medical Research Council Investigator Grants (GNT2009564 to D.J.C. and GNT2018461 to F.B.H.R.).
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F.B.H.R. conceptualized the work and led the individual studies detailed in the protocol. S.J.d.V., Y.Z., F.B.H.R. and T.D. performed experiments and analyzed data. T.D. and D.J.C. supervised the work. The manuscript was written by S.J.d.V. and Y.Z. with input from all authors.
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Nature Protocols thanks Yi-Ming Li, Yan-Mei Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references
de Veer, S. J. et al. J. Am. Chem. Soc. 147, 557–565 (2025): https://doi.org/10.1021/jacs.4c11964
Rehm, F. B. H. et al. Nat. Chem. 16, 1481–1489 (2024): https://doi.org/10.1038/s41557-024-01520-1
Rehm, F. B. H. et al. Angew. Chem. Int. Ed. 61, e202116672 (2022): https://doi.org/10.1002/anie.202116672
Rehm, F. B. H. et al. J. Am. Chem. Soc. 143, 19498–19504 (2021): https://doi.org/10.1021/jacs.1c08976
Yap, K. et al. Nat. Protoc. 16, 1740–1760 (2021): https://doi.org/10.1038/s41596-020-00483-0
This protocol is an extension to: Nat. Protoc. 16, 1740–1760 (2021): https://doi.org/10.1038/s41596-020-00483-0
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de Veer, S.J., Zhou, Y., Rehm, F.B.H. et al. Site-specific protein and peptide modification by redeploying an asparaginyl ligase for noncanonical reactions. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01348-8
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DOI: https://doi.org/10.1038/s41596-026-01348-8