Abstract
Tubulin tyrosine ligase (TTL) catalyzes the addition of tyrosine derivatives to the C-terminal carboxylic acid of proteins. The enzyme binds to a 14-amino acid recognition sequence, termed Tub-tag, and allows for the introduction of tyrosine derivatives that carry a unique chemical handle. These handles enable subsequent bioorthogonal reactions with a great variety of probes or effector molecules. Clearly, this two-step chemoenzymatic approach, facilitates the site-specific functionalization of proteins. Furthermore, due to its broad substrate tolerance, tubulin tyrosine ligase also enables an enzymatic one-step modification. For example, a coumarin amino acid was utilized to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics. Here we describe the modification of proteins using TTL in detail via a one-step as well as two-step procedure and highlight its practicability for applications in imaging, diagnostics, and cell biology.
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Acknowledgments
This work was supported by grants from the (Exist Forschungstransfer I), the Bavarian Ministry of Economic Affairs, Energy and Technology (m4-Award), the Deutsche Forschungsgemeinschaft (SPP1623) to C.P.R.H. (HA 4468/9-1) and H.L. (LE 721/13-1), the Nano-systems Initiative Munich (NIM) to H.L., the Einstein Foundation Berlin (Leibniz-Humboldt Professorship), the Boehringer-Ingelheim Foundation (Plus 3 award) to C.P.R.H. and from the Fonds der Chemischen Industrie (FCI) to C.P.R.H. and to D.S. (Kekulé-scholarship).
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Gerlach, M., Stoschek, T., Leonhardt, H., Hackenberger, C.P.R., Schumacher, D., Helma, J. (2019). Tubulin Tyrosine Ligase-Mediated Modification of Proteins. In: Nuijens, T., Schmidt, M. (eds) Enzyme-Mediated Ligation Methods. Methods in Molecular Biology, vol 2012. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9546-2_17
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DOI: https://doi.org/10.1007/978-1-4939-9546-2_17
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