Flavin-tag: exploiting a flavin transferase for protein labeling and engineering

Site-specific protein labeling plays a core role in investigating protein function at molecular level. A multitude of protein labelling techniques has been developed which are based on chemical and/or enzymatic methods. Enzyme-based labeling of proteins typically relies on a specific peptide sequence (a “tag”) that can be recognized and modified by a specific protein-modifying enzymes. In nature, some bacteria possess a flavin-transferase (called ApbE) which covalently incorporates an FMN cofactor in the so-called FMN-binding motif of some specific redox enzymes and proteins of which the role still has to be established. Based on this flavin transferase and its conserved recognition motif, we developed an enzymatic site-specific protein labelling method: the Flavin-tag method. This thesis involves the establishment, optimization, and application of the Flavin-tag method. We also expressed and characterized a multi-FMN-binding protein from Streptomyces azureus (SaFMN3) that is predicted to carry three covalently attached FMNs. This has unveiled some of the biochemical properties of such an extracellular multi-FMN-binding protein.