Expression, purification, and physicochemical characterization of theN‐terminal active site of human angiotensin‐I converting enzyme
作者:
Sotirios‐Spyridon M. Vamvakas,
Leondios Leondiadis,
George Pairas,
Evy Manessi‐Zoupa,
Georgios A. Spyroulias,
Paul Cordopatis,
期刊:
Journal of Peptide Science
(WILEY Available online 2007)
卷期:
Volume 13,
issue 1
页码: 31-36
ISSN:1075-2617
年代: 2007
DOI:10.1002/psc.788
出版商: John Wiley&Sons, Ltd.
关键词: angiotensin‐I converting enzyme;ACE;Renin‐angiotensin system;recombinant protein;zinc metallopeptidase
数据来源: WILEY
摘要:
AbstractWe have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361to Gly468, ACE‐N) of human somatic angiotensin‐I converting enzyme inEscherichia coli.This construct represents theN‐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‐Murea 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 W
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