Isolation of aminoglycoside nucleotidyltransferase (2'')-Ia from inclusion bodies as active, monomeric enzyme

Edward Wright; Engin H Serpersu

doi:10.1016/j.pep.2004.02.003

Isolation of aminoglycoside nucleotidyltransferase (2'')-Ia from inclusion bodies as active, monomeric enzyme

Protein Expr Purif. 2004 Jun;35(2):373-80. doi: 10.1016/j.pep.2004.02.003.

Authors

Edward Wright¹, Engin H Serpersu

Affiliation

¹ Center of Excellence in Structural Biology, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Walters Life Sciences Building M407, Knoxville, TN 37996-0840, USA.

PMID: 15135416
DOI: 10.1016/j.pep.2004.02.003

Abstract

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 0.1M Tris-HCl, pH 8.5, 0.2M KCl, 0.4M l-arginine, and 5mM reduced glutathione at 4 degrees C. The refolded enzyme is monomeric in solution and has similar kinetic properties and substrate selectivity to the enzyme isolated in soluble form.

Publication types

Research Support, U.S. Gov't, P.H.S.

MeSH terms

Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli / genetics
Inclusion Bodies / enzymology*
Nucleotidyltransferases / isolation & purification*
Substrate Specificity

Substances

Nucleotidyltransferases