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Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry

Abstract

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.

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Figure 1: Illustrative overview of the protocol.
Figure 2: Synthesis of AzC12.
Figure 3: Synthesis of trifunctional capture reagent.
Figure 4
Figure 5: Typical results.
Figure 6: Troubleshooting gels (gel used for illustrative purposes only).

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Acknowledgements

We are grateful to the UK Biotechnology and Biological Sciences Research Council (grant BB/D02014X/1 to E.W.T.), to the UK Engineering and Physical Sciences Research Council and the Imperial College Institute of Chemical Biology (PhD studentship to M.H.W.), and to Cancer Research UK (grant C29637/A 10711 to E.W.T.) for funding this work.

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W.P.H. conducted the initial syntheses; carried out the bacterial, click chemistry and SDS-PAGE/imaging work; and prepared the first draft of the manuscript and subsequent modifications. M.H.W. refined the design and synthesis of the trifunctional capture reagent and optimized the click chemistry conditions. E.T. refined the synthesis of and prepared the azido-myristate analog, and was also involved in manuscript preparation. E.W.T. supervised all of the work and oversaw preparation of the final version of the manuscript.

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Correspondence to Edward W Tate.

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Heal, W., Wright, M., Thinon, E. et al. Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry. Nat Protoc 7, 105–117 (2012). https://doi.org/10.1038/nprot.2011.425

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