Over-expression, Purification, and Characterization of Metallo-Beta-Lactamase ImiS From Aeromonas Veronii Bv. Sobria

Protein Expr Purif. 2004 Aug;36(2):272-9. doi: 10.1016/j.pep.2004.04.017.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Chromatography, Ion Exchange
  • Circular Dichroism
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Kinetics
  • Molecular Weight
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Substrate Specificity
  • Zinc / chemistry*
  • beta-Lactamases / chemistry*
  • beta-Lactamases / genetics
  • beta-Lactamases / isolation & purification*

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • ImiS protein, Aeromonas veronii
  • beta-Lactamases
  • Zinc