Over-Expression, Purification and Characterization of Metallo-beta-Lactamase ImiS from Aeromonas veronii bv. sobria

PA Crawford, N Sharma, S Chandrasekar, T Sigdel, TR Walsh, J Spencer, MW Crowder

Research output: Contribution to journalArticle (Academic Journal)peer-review

42 Citations (Scopus)

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 Km 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.
Translated title of the contributionOver-Expression, Purification and Characterization of Metallo-beta-Lactamase ImiS from Aeromonas veronii bv. sobria
Original languageEnglish
Pages (from-to)272 - 279
Number of pages8
JournalProtein Expression and Purification
Volume36
DOIs
Publication statusPublished - Aug 2004

Bibliographical note

Publisher: Elsevier

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