Abstract
Large amounts of heterologous C-terminally his-tagged SERCA1a Ca 2+-ATPase were expressed in yeast using a galactose-regulated promoter and purified by Ni 2+ affinity chromatography followed by Reactive red chromatography. Optimizing the number of galactose inductions and increasing the amount of Gal4p transcription factor improved expression. Lowering the temperature from 28°C to 18°C during expression enhanced the recovery of solubilized and active Ca 2+-ATPase. In these conditions, a 4 l yeast culture produced 100 mg of Ca 2+-ATPase, 60 and 22 mg being pelleted with the heavy and light membrane fractions respectively, representing 7 and 1.7% of total proteins. The Ca 2+-ATPase expressed in light membranes was 100% solubilized with L-α-lysophosphatidylcholine (LPC), 50% with n-dodecyl β- D-maltoside (DM) and 25% with octaethylene glycol mono- n-dodecyl ether (C 12E 8). Compared to LPC, DM preserved specific activity of the solubilized Ca 2+-ATPase during the chromatographic steps. Starting from 1/6 (3.8 mg) of the total amount of Ca 2+-ATPase expressed in light membranes, 800 μg could be routinely purified to 50% purity by metal affinity chromatography and then 200 μg to 70% with Reactive red chromatography. The purified Ca 2+-ATPase displayed the same K m for calcium and ATP as the native enzyme but a reduced specific activity ranging from 4.5 to 7.3 μmol ATP hydrolyzed/min/mg Ca 2+-ATPase. It was stable and active for several days at 4°C or after removal of DM with Bio-beads and storage at −80°C.
Published Version
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