[en] The Aeromonas hydrophila CphA metallo-beta-lactamase was overexpressed in a soluble secreted form in Escherichia coli using a T7 RNA polymerase-based expression system, and a simple protocol based on a single cation-exchange chromatographic step was developed, which is suitable for rapid purification of the overexpressed enzyme from E. coli lysates. A yield of up to 30 micrograms of purified enzyme per milliliter of culture was obtained. The purified enzyme preparation showed properties identical to those previously reported in the literature.
Ambler, R.P., The structure of β-lactamases (1980) Philos. Trans. R. Soc. London Sect. B Biol. Sci., 289, pp. 321-331
Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding (1976) Anal. Biochem., 72, pp. 248-254
Felici, A., Amicosantc, G., Oratore, A., Strom, R., Ledent, P., Joris, B., Fanuel, L., Frère, J.-M., An overview of the kinetic parameters of class B β-lactamases (1993) Biochem. J., 291, pp. 151-155
Frère, J.-M., Beta-lactamases and bacterial resistance to antibiotics (1995) Mol. Microbiol., 16, pp. 385-395
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature (London), 227, pp. 680-685
Massidda, O., Rossolini, G.M., Satta, G., The Aeromonas hydrophila cphA gene: Molecular heterogeneity among class B metallo β-lactamases (1991) J. Bacteriol., 173, pp. 4611-4617
Sambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schein, C.H., Solubility as a function of protein structure and solvent components (1990) Bio/Technology, 8, pp. 308-317
Segatore, B., Massidda, O., Satta, G., Setacci, D., Amicosante, G., High specificity of cphA-encoded metallo-β-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to β-lactam resistance (1993) Antimicrob. Agents Chemother., 37, pp. 1324-1328
Segel, L.H., (1976) Biochemical Calculations, , John Wiley, New York
Studier, F.W., Rosenberg, A.H., Dunn, J.J., Dubendorff, J.W., Use of T7 RNA polymerase to direct expression of cloned genes (1990) Methods Enzymol., 185, pp. 60-89
