Source:http://linkedlifedata.com/resource/pubmed/id/16380266
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
2
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pubmed:dateCreated |
2006-5-23
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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.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Aminopeptidases,
http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Enzyme Inhibitors,
http://linkedlifedata.com/resource/pubmed/chemical/Iron,
http://linkedlifedata.com/resource/pubmed/chemical/Zinc,
http://linkedlifedata.com/resource/pubmed/chemical/pepN protein, Bacteria
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pubmed:status |
MEDLINE
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pubmed:month |
Jun
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pubmed:issn |
1046-5928
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:volume |
47
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
634-9
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:16380266-Aminopeptidases,
pubmed-meshheading:16380266-Bacterial Proteins,
pubmed-meshheading:16380266-Chromatography, Gel,
pubmed-meshheading:16380266-Enzyme Inhibitors,
pubmed-meshheading:16380266-Escherichia coli,
pubmed-meshheading:16380266-Gene Expression,
pubmed-meshheading:16380266-Iron,
pubmed-meshheading:16380266-Kinetics,
pubmed-meshheading:16380266-Protein Binding,
pubmed-meshheading:16380266-Spectrometry, Mass, Matrix-Assisted Laser...,
pubmed-meshheading:16380266-Substrate Specificity,
pubmed-meshheading:16380266-Zinc
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pubmed:year |
2006
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pubmed:articleTitle |
Over-expression, purification, and characterization of aminopeptidase N from Escherichia coli.
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pubmed:affiliation |
Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, USA.
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pubmed:publicationType |
Journal Article,
Research Support, N.I.H., Extramural
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