A VERSATILE PLASMID EXPRESSION VECTOR FOR THE PRODUCTION OF BIOTINYLATED PROTEINS BY SITE-SPECIFIC, ENZYMATIC MODIFICATION IN ESCHERICHIA-COLI

A versatile plasmid vector was designed to direct the synthesis of rec ombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. Th e protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N term inus, or alternatively, as a fusion to the C terminus of a maltose-bin ding protein domain (MalE) with the peptide substrate on its N terminu s, To maximize the yield of biotinylated protein, the vector is design ed to express the substrate in a coupled translation arrangement with the enzyme.