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Creating Site-Specific Isopeptide Linkages Between Proteins with the Traceless Staudinger Ligation

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Book cover Peptide Libraries

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1248))

Abstract

Site-specific isopeptide linkages between the ε-amino group of a lysine residue in one protein and a carboxyl group in another are central to ubiquitin-mediated protein degradation and other cellular processes. These linkages are inaccessible with common recombinant DNA techniques. Here, we describe a method to link two proteins by an authentic isopeptide bond. The method unites three techniques at the forefront of molecular biology. An azidonorleucine residue is installed at a desired site in a substrate protein by nonnatural amino acid incorporation, and a phosphinothioester is installed at the C terminus of a pendant protein by expressed protein ligation. Then, the traceless Staudinger ligation is used to link the substrate and pendant proteins via an isopeptide bond. This method facilitates the study of otherwise intractable protein structure–function relationships.

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Acknowledgements

We are grateful to Drs. L. J. Martin and I. C. Tanrikulu for contributive discussions. This work was supported by Grant R01 GM044783 (NIH). K.A.A. was supported by the Molecular and Cellular Pharmacology Training Grant T32 GM008688 (NIH) and a predoctoral fellowship from the PhRMA Foundation.

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin – Madison, 433 Babcock Drive, Madison, WI, 53706-1544, USA

    Kristen A. Andersen & Ronald T. Raines

Authors
  1. Kristen A. Andersen
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  2. Ronald T. Raines
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Correspondence to Ronald T. Raines .

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Editors and Affiliations

  1. Dept of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Ratmir Derda

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Andersen, K.A., Raines, R.T. (2015). Creating Site-Specific Isopeptide Linkages Between Proteins with the Traceless Staudinger Ligation. In: Derda, R. (eds) Peptide Libraries. Methods in Molecular Biology, vol 1248. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2020-4_4

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  • DOI: https://doi.org/10.1007/978-1-4939-2020-4_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2019-8

  • Online ISBN: 978-1-4939-2020-4

  • eBook Packages: Springer Protocols

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