Folding in solution of the C-catalytic protein fragment of angiotensin-converting enzyme.
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 Ala(959) to Ser(1066) 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 ACE(959-1066) protein fragment in order to study its structure in solution by NMR spectroscopy.
Vamvakas
Sotirios-Spyridon M
SS
Mass Spectrometry and Dioxin Analysis Lab, IRRP, National Center for Scientific Research "Demokritos", 27 Neapoleos St., GR-15310 Athens, Greece.
Leondiadis
Leondios
L
Pairas
George
G
Manessi-Zoupa
Evy
E
Spyroulias
Georgios A
GA
Cordopatis
Paul
P
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012996:Solutions / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D007527:Isoenzymes; D010446:Peptide Fragments; D012996:Solutions; D007703:Peptidyl-Dipeptidase A