Linked Life Data

15249050

Source:http://linkedlifedata.com/resource/pubmed/id/15249050

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2004-7-13
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
272-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
Over-expression, purification, and characterization of metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria.
pubmed:affiliation
Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, OH 45056, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't