Folding in solution of the C‐catalytic protein fragment of angiotensin‐converting enzyme |
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Authors: | Sotirios‐Spyridon M. Vamvakas Leondios Leondiadis George Pairas Evy Manessi‐Zoupa Georgios A. Spyroulias Paul Cordopatis |
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Affiliation: | 1. Mass Spectrometry and Dioxin Analysis Lab, IRRP, National Center for Scientific Research “Demokritos”, 27 Neapoleos St., GR‐15310 Athens, Greece;2. Department of Pharmacy, University of Patras, GR‐26500 Patras, Greece;3. Department of Chemistry, University of Patras, GR‐26500 Patras, Greece |
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Abstract: | Angiotensin‐converting enzyme (ACE) is a key molecule of the renin–angiotensin–aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala959 to Ser1066 region (ACE_C) that corresponds to the C‐catalytic domain of human somatic angiotensin‐I‐converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1‐trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE959–1066 protein fragment in order to study its structure in solution by NMR spectroscopy. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. |
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Keywords: | ACE zinc metallopeptidase recombinant protein secondary structure circular dichroism |
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