Microbial Transglutaminase and c‐myc‐Tag: A Strong Couple for the Functionalization of Antibody‐Like Protein Scaffolds from Discovery Platforms

Abstract Antibody‐like proteins selected from discovery platforms are preferentially functionalized by site‐specific modification as this approach preserves the binding abilities and allows a side‐by‐side comparison of multiple conjugates. Here we present an enzymatic bioconjugation platform that targets the c‐myc‐tag peptide sequence (EQKLISEEDL) as a handle for the site‐specific modification of antibody‐like proteins. Microbial transglutaminase (MTGase) was exploited to form a stable isopeptide bond between the glutamine on the c‐myc‐tag and various primary‐amine‐functionalized substrates. We attached eight different functionalities to a c‐myc‐tagged antibody fragment and used these bioconjugates for downstream applications such as protein multimerization, immobilization on surfaces, fluorescence microscopy, fluorescence‐activated cell sorting, and in vivo nuclear imaging. The results demonstrate the versatility of our conjugation strategy for transforming a c‐myc‐tagged protein into any desired probe.