Two‐Step Protein Labeling by Using Lipoic Acid Ligase with Norbornene Substrates and Subsequent Inverse‐Electron Demand Diels–Alder Reaction |
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Authors: | Marcel Best Anna Degen Mathis Baalmann Tobias T. Schmidt Dr. Richard Wombacher |
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Affiliation: | Institute for Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg (Germany) |
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Abstract: | Inverse‐electron‐demand Diels–Alder cycloaddition (DAinv) between strained alkenes and tetrazines is a highly bio‐orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two‐step labeling protocol for the site‐specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplAW37V), followed by the covalent attachment of tetrazine‐modified fluorophores to the norbornene moiety through the bio‐orthogonal DAinv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13‐residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two‐step labeling strategy to label proteins in cell lysates in a site‐specific manner and performed cell‐surface labeling on living cells. |
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Keywords: | fluorescent probes inverse‐electron‐demand Diels– Alder cycloaddition ligases protein labeling tetrazines |
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