Transglutaminase-mediated internal protein labeling with a designed peptide loop

► A 13-mer substrate peptide was designed for microbial transglutaminase (MTG). ► The substrate peptide was internally inserted into alkaline phosphatase (AP). ► Random coil regions of AP were selected for peptide insertion. ► A lysine in the inserted loop was site-specifically labeled by MTG. ► Enzymatic internal protein labeling was achieved without loss of AP activity.