Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE)

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-HCI buffer (50 mM, pH 9.0) for 48 h. An automation of the ACE purification was performed using a multi-step protocol in less than 8 h, resulting in a purified protein with a specific activity of 37 U mg(-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 (TM) 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 similar to 175 kDa by SDS-PAGE, with the dimeric form at similar to 330 kDa 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 H2O2 stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks. (C) 2012 Elsevier B.V. All rights reserved.