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Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry

Nature Protocols volume 8, pages 1808–1819 (2013)Cite this article

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Abstract

Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3–4 d.

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Figure 1: Schematic representation of the strategy to produce unnaturally linked C-to-C and N-to-N chimeric proteins.
Figure 2
Figure 3: Typical results of sortagging and subsequent click reactions.

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Acknowledgements

This work was supported by funding from The Netherlands Organisation for Scientific Research (to M.D.W.) and the US National Institutes of Health (NIH; grant no. RO1 AI087879 to H.L.P.).

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Author notes

  1. Martin D Witte

    Present address: Present address: Bio-Organic Chemistry, Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands.,

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Martin D Witte, Christopher S Theile, Carla P Guimaraes, Annet E M Blom & Hidde L Ploegh

  2. Oncology Medicinal Chemistry, Janssen Research and Development, Beerse, Belgium

    Tongfei Wu

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Hidde L Ploegh

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  1. Martin D Witte
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Contributions

M.D.W. and H.L.P. conceived of and drafted the manuscript; M.D.W., C.S.T., T.W. and C.P.G. participated in the optimization of the protocols and wrote the manuscript. A.E.M.B. and H.L.P. revised the manuscript.

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Correspondence to Hidde L Ploegh.

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Competing interests

The authors declare that a patent application (no. WO2013003555 A1) on the production of N-to-N and C-to-C fusions has been filed.

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Witte, M., Theile, C., Wu, T. et al. Production of unnaturally linked chimeric proteins using a combination of sortase-catalyzed transpeptidation and click chemistry. Nat Protoc 8, 1808–1819 (2013). https://doi.org/10.1038/nprot.2013.103

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