BirA enzyme: Production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation

The enzyme BirA is a key reagent because of its ability to biotinylate prot eins at a specific residue in a recognition sequence. We report a rapid, ef ficient, and economical method for the production, purification, and applic ation of this enzyme, The method is easily scaled up and the protein produc ed is of high purity and can be stored for many months with retention of ac tivity. We have used this enzyme to biotinylate the C termini of membrane p roteins, allowing these proteins to be tetramerized by binding to streptavi din, Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface. These tetrameric proteins c an be used to study protein receptor-ligand interactions at the cell surfac e, and site-specific biotinylation can be used to study proteins in vitro u sing a defined orientation. (C) 1999 Academic Press.