Simple azide labeling of biotin-binding proteins using microbial transglutaminase: A practical note

Microbial transglutaminase (MTG) is a Ca²⁺-independent enzyme that enables site-specific protein labeling under mild conditions. However, how substrate structure and protein-specific environments influence MTG reactivity is not yet fully understood. In this study, we synthesized a novel glutamine-based azide donor (compound 1) and used it alongside a commercially available amine-based azide acceptor (3-azido-1-propylamine) to evaluate MTG-mediated labeling of five biotin-binding proteins: Avidin, Streptavidin, NeutrAvidin, Tamavidin 2, and Tamavidin 2-LPI. Azide-modified proteins were visualized using strain-promoted azide–alkyne cycloaddition (SPAAC) with DBCO-Cy5, and labeling efficiency was assessed by comparing MTG+ and MTG− conditions. Tamavidin 2 showed high reactivity toward both substrates, while Tamavidin 2-LPI exhibited little to no labeling. Streptavidin was labeled only by the amine-based substrate, whereas Avidin and NeutrAvidin were mainly labeled by the glutamine-based substrate. Notably, NeutrAvidin also showed strong fluorescence in the absence of MTG, suggesting non-specific interaction. These results indicate that MTG-mediated labeling is governed by both substrate compatibility and protein-dependent factors. Although the exact modification sites remain unidentified, this study provides a practical framework for selective biotin-protein conjugation using MTG and complementary azide reagents.