A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein...

Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins. The data obtained in this study reveal how biotin and ATP significantly increase the thermal stability (ΔT_m=+16.5°C) of M. tuberculosis BirA and lead to formation of a high affinity holoenzyme complex (K_obs=7.7nM). The new findings and mycobacterial BirA high-throughput assay presented in this work could provide an efficient platform for future anti-tubercular drug discovery campaigns.