Tetracycline (Tc) is a broad spectrum antibiotic that binds to the A site of the bacterial ribosome inhibiting delivery of aminoacyl-tRNA to the A site for productive protein biosynthesis. Tet(O) is in a class of the ribosomal protection proteins (RPPs) found in many pathogenic bacteria, that dislodges Tc from the A site of 70S ribosome to restore polypeptide elongation and confer Tc resistance to the bacteria. Considerable difficulty has been encountered in overexpressing and purifying Tet(O) from various Escherichia coli strains using),lambda PI, tac or T7 promoters. Here we report molecular cloning, overexpression of His-tagged Tet(O) in E coli, an improved purification procedure and initial biochemical and biophysical characterization of His-tagged Tet(O). (C) 2007 Elsevier Inc. All rights reserved.