Expression, purification, and physicochemical characterization of the N-terminal active site of human angiotensin-I converting enzyme.
We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361 to Gly468, 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.
Vamvakas
Sotirios-Spyridon M
SS
Laboratory of Mass Spectrometry and Dioxin Analysis, National Center for Scientific Research Demokritos, 27 Neapoleos Str, GR-15310 Athens, Greece.
Leondiadis
Leondios
L
Pairas
George
G
Manessi-Zoupa
Evy
E
Spyroulias
Georgios A
GA
Cordopatis
Paul
P
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D007703:Peptidyl-Dipeptidase A