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

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.