Broadening the Scope of the Flavin‐Tag Method by Improving Flavin Incorporation and Incorporating Flavin Analogs

Abstract

Methods for facile site-selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site-specific covalent attachment of a chromoand fluorogenic flavin (FMN) to any targeted protein. Although this Flavin-tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin-tag method that addresses previous limitations. We demonstrate that co-expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin-labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor

“…Yet, for variant F1, MS analysis revealed that part of the protein did not contain covalent FMN (Figure b). Previous studies showed that culturing conditions (temperature and riboflavin concentration) could influence the flavinylation of the target protein containing a Flavin-tag . Therefore, we optimized the induction temperature and riboflavin concentration to boost the incorporation of covalent FMN into PpSB1-LOV F1.…”

“…Previous work has shown that coexpression of both enzymes boosts the levels of flavin incorporation into the Flavin-tag. 14 For the coexpression experiments, the plasmid pRSF-Duet1 (expressing ApbE and CaFADS) was cotransformed with the plasmid carrying a PpSB1-LOV-encoding gene. As a reference, wildtype PpSB1-LOV was also coexpressed with the flavinylation machinery and purified.…”

“…We have recently shown that the above-mentioned flavin transferase can be used for directed labeling of target proteins with a covalent flavin, a flavin analogue, or even a nicotinamide group. By equipping a protein with an N- or C-terminal peptide that includes a flavinylation recognition site (DxxxGA­[ T / S ], flavinylated amino acid in bold), FMN can be enzymatically attached using a truncated, and therefore soluble, version of the bacterial flavin transferase from Vibrio cholerae (ApbE). − We termed this approach Flavin-tagging and have successfully applied it for labeling several proteins with FMN or FMN derivatives with altered chromogenic, fluorescence, and redox properties. , The Flavin-tag system can be used as a convenient tool for decorating a target protein with a flavin-based probe. Selective covalent labeling can be achieved in vivo (by coexpressing the target protein and the flavin transferase) and in vitro (by incubating the target protein with the flavin transferase and FAD).…”

“…11−13 We termed this approach Flavin-tagging and have successfully applied it for labeling several proteins with FMN or FMN derivatives with altered chromogenic, fluorescence, and redox properties. 13,14 The Flavin-tag system can be used as a convenient tool for decorating a target protein with a flavin-based probe. Selective covalent labeling can be achieved in vivo (by coexpressing the target protein and the flavin transferase) and in vitro (by incubating the target protein with the flavin transferase and FAD).…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…This method facilitates purification and detection due to the fluorescence and yellow color of the flavin‐labelled proteins [187] . The presence of FAD synthetase improves the efficiency of flavin incorporation and was used to label alcohol dehydrogenase, maltose‐binding protein, and ubiquitinin‐related SUMO [188] . ApbE accepts FMN‐derivatives as well as nicotinamide and the bright‐red, roseoflavin compound (Figure 9ii).…”