Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1337; p. 109
• Main Authors: Wagner, Anne M, Warner, John B, Garrett, Haviva E, Walters, Christopher R, Petersson, E James
• Format: Journal Article
• Language: English
• Published: United States 2015
• Subjects: Aminoacyltransferases - metabolism; Arginine-tRNA Ligase - metabolism; Catalysis; Click Chemistry - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Protein Interaction Domains and Motifs; Proteins - chemistry; Proteins - metabolism; Recombinant Proteins; Staining and Labeling; Transferases - metabolism
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-2935-1_15

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This method can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.