Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2006-5-23
pubmed:abstractText
The gene from Escherichia coli encoding aminopeptidase N (PepN) was subcloned into pET-26b, and PepN was over-expressed in BL21(DE3) E. coli and purified using Q-Sepharose chromatography. This protocol yielded over 17 mg of purified, recombinant PepN per liter of growth culture under optimum conditions. Gel filtration chromatography revealed that recombinant PepN exists as a monomer. MALDI-TOF mass spectra showed that the enzyme has a molecular mass of 98,750 Da, and steady-state kinetic studies revealed that as-isolated, recombinant PepN exhibits a k(cat) of 354 +/- 11s(-1) and a K(m) of 376 +/- 39 microM when using L-alanine-p-nitroanilide as the substrate. Metal analyses demonstrated that as-isolated, recombinant PepN binds 0.5 and <0.1 equivalents of iron and zinc, respectively. The addition of Zn(II) to recombinant PepN inhibits catalytic activity, while the addition of iron causes a slight decrease or no change in activity. Further metal binding studies revealed that recombinant PepN tightly binds 5 equivalents of iron and <0.1 equivalents of Zn(II). By using this over-expression and purification system, E. coli PepN can now be obtained in quantities necessary for structural characterization and possibly inhibitor design efforts.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
47
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
634-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Over-expression, purification, and characterization of aminopeptidase N from Escherichia coli.
pubmed:affiliation
Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural