Over-expression, purification, and characterization of aminopeptidase N from Escherichia coli.
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.
Golich
Frank C
FC
Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, OH 45056, USA.
Han
Maria
M
Crowder
Michael W
MW
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D015870:Gene Expression; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C075633:pepN protein, Bacteria; D007501:Iron; D000626:Aminopeptidases; D015032:Zinc