BirA, the most characterized biotin-acetyl-CoA-carboxylase ligase, has been successfully applied to biotin labeling with a wide range of protein substrates that fused an acceptor peptide （AP, also known as Avitag） in vivo and in vitro. In the presence of ATP, BirA activates biotin to form the BirA-biotinyl-5'-adenylate （BirA-bio-5'-AMP or holoBirA） complex. BirA can either transfer the biotinyl moiety to the biotin carboxyl carrier protein （BCCP） subunit of acetyl-CoA carboxylase. Due to its extremely high strength, the interaction between biotin and streptavidin has been exploited for biotechnological applications, like in vivo imaging, cell sorting, et al.