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.