Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

Abstract

In E. coli and other prokaryotes such as Staphylococcus aureus, and Mycobacterium tuberculosis, biotin protein ligase (BirA) is an emerging drug target as it is the sole enzyme capable of biotin transfer onto the BCCP subunit of ACC. There is currently a gap in simple yet efficient assays for rapidly identifying and characterising inhibitors targeting BirA. We present for the first time the development and validation of a simple and reliable DSF-GTP assay for the high-throughput screening of BirA:ligand interactions using a new GFP-tagged BirA of E. coli. In addition, we developed a new GFP-based biotinylation activity assay taking advantage of a GFP tethered with an AviTag. The data obtained with these assays revealed new insights into how the binding of individual or combinations of ligands affect the overall thermal stability and affinity of BirA. The DSF-GTP assay has a Z′ value of 0.785 that makes it an excellent tool for future high-throughput screening of inhibitory compounds.