title: Over-expression, purification, and characterization of metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria.
abstract: The gene from Aeromonas veronii bv. sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography. This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions. The biochemical properties of recombinant ImiS were compared with those of native ImiS. Recombinant and native ImiS have the same N-terminus of A-G-M-S-L, and CD spectroscopy was used to show that the enzymes have similar secondary structures. Gel filtration chromatography revealed that both enzymes exist as monomers in solution. MALDI-TOF mass spectra showed that the enzymes have a molecular mass of 25,247 Da, and metal analyses demonstrated that both as-isolated enzymes bind ca. 0.7 mol of Zn(II). Metal titrations demonstrate that the maximum activity of recombinant ImiS occurs when the enzyme binds one equivalent of zinc. Steady-state kinetic studies reveal that recombinant ImiS is a carbapenemase like native ImiS and that the recombinant enzyme exhibits similar kcat and K(m) values for the substrates tested, as compared to the native enzyme. This over-expression protocol now allows for detailed spectroscopic and mechanistic studies on ImiS as well as site-directed mutants of ImiS to be prepared for future structure/function studies.
authors.affiliation: Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, OH 45056, USA.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Crawford
authors.forename: Narayan
authors.initials: N
authors.lastname: Sharma
authors.forename: Sowmya
authors.initials: S
authors.lastname: Chandrasekar
authors.forename: Tara
authors.initials: T
authors.lastname: Sigdel
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Walsh
authors.forename: James
authors.initials: J
authors.lastname: Spencer
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C111082:ImiS protein, Aeromonas veronii; D001618:beta-Lactamases; D015032:Zinc