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Expression, purification, and physicochemical characterization of the N ‐terminal active site of human angiotensin‐I converting enzyme
Author(s) -
Vamvakas SotiriosSpyridon M.,
Leondiadis Leondios,
Pairas George,
ManessiZoupa Evy,
Spyroulias Georgios A.,
Cordopatis Paul
Publication year - 2007
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.788
Subject(s) - chemistry , inclusion bodies , escherichia coli , recombinant dna , enzyme , peptide , size exclusion chromatography , biochemistry , urea , amino acid , peptide sequence , nuclear magnetic resonance spectroscopy , chromatography , stereochemistry , gene
We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala 361 to Gly 468 , ACE‐N) of human somatic angiotensin‐I converting enzyme in Escherichia coli. This construct represents the N ‐catalytic domain including the two binding motifs and the 23 amino acid spacers as well as some amino acid residues before and after the motifs that might help in correct conformation. The overexpressed protein was exclusively localized to insoluble inclusion bodies. Inclusion bodies were solubilized in an 8‐ M urea buffer. Purification was carried out by differential centrifugation and gel filtration chromatography under denaturing conditions. About 12 mg of ACE‐N peptide per liter of bacterial culture was obtained. The integrity of recombinant protein domain was confirmed by ESI/MS. Structural analysis using CD spectroscopy has shown that, in the presence of TFE, the ACE‐N protein fragment has taken a conformation, which is consistent with the one found in testis ACE by X‐ray crystallography. This purification procedure enables the production of an isotopically labeled protein fragment for structural studying in solution by NMR spectroscopy. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.