Overproduction and purification of the Aeromonas hydrophila CphA metallo-beta-lactamase expressed in Escherichia coli.
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.
Hernandez Villadares
M
M
Dipartimento di Scienze e Tecnologie Biomediche e di Biometria, Università dell'Aquila, Italy.
Galleni
M
M
Frère
J M
JM
Felici
A
A
Perilli
M
M
Franceschini
N
N
Rossolini
G M
GM
Oratore
A
A
Amicosante
G
G
D016980:Aeromonas hydrophila / Q000201:enzymology* / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005822:Genetic Vectors; D006868:Hydrolysis; D015378:Imipenem / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D015378:Imipenem; D001618:beta-Lactamases; C082063:cphA protein, Aeromonas hydrophila