Phage-Selected Clickable Gln-Donor Peptide for Lys-Selective Fab Labeling Using Engineered Microbial Transglutaminase

Background/Objectives: The use of cross-linking enzymes for site-selective and efficient antibody modification has attracted considerable attention. Microbial transglutaminase (MTG)-mediated labeling of IgG at Gln295 has emerged as a promising strategy for preparing antibody–drug conjugates (ADCs). By contrast, selective modification of a specific Lys residue on native antibody surfaces using MTG remains challenging because most Lys residues exhibit low intrinsic reactivity. Here, we address this challenge by exploiting enzyme–antibody proximity together with screening for highly reactive Gln-donor substrates from a random peptide library. Methods: Reactive Gln-donor peptide substrates were first identified from a seven-amino-acid phage-displayed peptide library using a reactive Lys-containing peptide as bait. Based on the obtained sequence, an azide-functionalized Gln-donor peptide suitable for click chemistry was designed. Results: The designed substrate enabled efficient Lys65-selective modification of Fab fragments using a fusion of an engineered MTG zymogen and protein G (EzMTG-pG), followed by functionalization through click chemistry to yield fluorescent Fab conjugates. Conclusions: These results provide practical guidelines for substrate design in MTG-mediated site-selective protein modification.