Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation

► A novel protocol was developed for the production of BirA ligase in E. coli. ► The target protein was expressed as both thioredoxin and MBP fusions and released by TEV protease cleavage. ► Protein yield was significantly improved compared with that of previously reported non-fusion expression. ► The produced recombinant BirA is highly active in catalyzing in vitro biotinylation.