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Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation

Nature Protocols volume 10, pages 1594–1611 (2015)Cite this article

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Abstract

Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for CuI-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a 'bifunctional' protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.

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Figure 1: Overview of accessible protein-protein conjugates using this protocol.
Figure 2: Incorporation of unnatural amino acids Aha and Plk in nascent proteins.
Figure 3: Maps of vectors used in this protocol and scheme of GST-Ub fusion.
Figure 4: SDS-PAGE analysis of the click reactions for purified Ub G76Aha, Ub K11Plk G76Aha and Pol beta K61Plk.
Figure 5: Experimental guidelines for the generation of functionalized proteins.

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Acknowledgements

We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft within the SFB 969 'Chemical and Biological Principles of Cellular Proteostasis' and the SPP 1623, as well as the Carl Zeiss Stiftung (stipend to T.S.). We also acknowledge the Konstanz Research School Chemical Biology for support.

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  1. Daniel Rösner, Tatjana Schneider and Daniel Schneider: These authors contributed equally to this work.

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  1. Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

    Daniel Rösner, Tatjana Schneider, Daniel Schneider & Andreas Marx

  2. Department of Biology, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

    Daniel Schneider & Martin Scheffner

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Contributions

A.M., M.S., D.R., T.S. and D.S. designed the research. D.R., T.S. and D.S. expressed and purified recombinant proteins and performed the experiments. A.M., M.S., D.R., T.S. and D.S. wrote the manuscript.

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Correspondence to Martin Scheffner or Andreas Marx.

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Rösner, D., Schneider, T., Schneider, D. et al. Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation. Nat Protoc 10, 1594–1611 (2015). https://doi.org/10.1038/nprot.2015.106

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