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

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 (Kobs =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.
Source: Microbiological Research - Category: Infectious Diseases Source Type: research