| Autor: |
Gerlach M; Department of Biology II, LMU Munich, Planegg/Martinsried, Germany., Stoschek T; Department of Biology II, LMU Munich, Planegg/Martinsried, Germany., Leonhardt H; Department of Biology II, LMU Munich, Planegg/Martinsried, Germany., Hackenberger CPR; Department of Chemical-Biology, Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.; Department of Chemistry, Humboldt Universität zu Berlin, Berlin, Germany., Schumacher D; Department of Biology II, LMU Munich, Planegg/Martinsried, Germany. schumacher@biologie.uni-muenchen.de.; Department of Chemical-Biology, Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany. schumacher@biologie.uni-muenchen.de., Helma J; Department of Biology II, LMU Munich, Planegg/Martinsried, Germany. helma@biologie.uni-muenchen.de. |
| Abstrakt: |
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. |
