期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 47, 期 2, 页码 634-639出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2005.11.012
关键词
aminopeptidase N; iron
类别
资金
- NIGMS NIH HHS [GM40052] Funding Source: Medline
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 17mg 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 +/- 11 s(-1) and a K-m of 376 +/- 39 mu M 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. (c) 2005 Elsevier Inc. All rights reserved.
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