Aminoglycoside nucleotidyltransferase(2")-Ia (ANT(2")) confers resistance to pathogenic bacteria against several aminoglycoside antibiotics including gentamicin, kanamycin, and tobramycin. The gene for this aminoglycoside-modifying enzyme has been cloned from a clinical isolate of Pseudomonas aeruginosa. This gene was inserted into an overexpression vector, the vector was then transformed into Escherichia coli BL21(DE3), and the protein has been isolated in the form of inclusion bodies. Optimal refolding conditions have been determined to be direct dilution of solubilized inclusion bodies into 0.1 M Tris-HCI, pH 8.5, 0.2 M KCl, 0.4 M L-arginine, and 5 mM reduced glutathione at 4 degreesC. The refolded enzyme is monomeric in solution and has similar kinetic properties and substrate selectivity to the enzyme isolated in soluble form. (C) 2004 Elsevier Inc. All rights reserved.