Two-Step Protein Labeling by Using Lipoic Acid Ligase with Norbornene Substrates and Subsequent Inverse-Electron Demand Diels-Alder Reaction

摘要

Inverse-electron-demand Diels-Alder cycloaddition (DA(inv)) 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 (LplA(W37V)), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DA(inv). 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.