title: Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE).
abstract: Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris-HCl buffer (50mM, pH 9.0) for 48h. An automation of the ACE purification was performed using a multi-step protocol in less than 8h, resulting in a purified protein with a specific activity of 37Umg(-1) (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175kDa by SDS-PAGE, with the dimeric form at ∼330kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H(2)O(2) stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks.
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology, Garbenstr. 25, 70599 Stuttgart, Germany. t.eisele@uni-hohenheim.de
authors.forename: Thomas
authors.initials: T
authors.lastname: Eisele
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.forename: Bertolt
authors.initials: B
authors.lastname: Kranz
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D000818:Animals; D001331:Automation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008168:Lung / Q000737:chemistry / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D013552:Swine
chemical_list: D009842:Oligopeptides; C043963:phenylalanyl-glycyl-glycine; D007703:Peptidyl-Dipeptidase A