Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca2+-ATPase

Large amounts of heterologous C-terminally his-tagged SERCAla Ca2+-ATPase were expressed in yeast using a galactose-regulated promoter and purified by Ni2+ affinity chromatography followed by Reactive red chromatography. Optimizing the number of galactose inductions and increasing the amount of Ga14p transcription factor improved expression. Lowering the temperature from 28degreesC to 18degreesC during expression enhanced the recovery of solubilized and active Ca2+-ATPase. In these conditions, a 41 yeast culture produced 100 mg of Ca2+-ATPase, 60 and 22 mg being pelleted with the heavy and light membrane fractions respectively, representing 7 and 1.7% of total proteins. The Ca2+-ATPase expressed in light membranes was 100% solubilized with L-alpha-lysophosphatidylcholine (LPC), 50% with n-dodecyl beta-D-maltoside (DM) and 25% with octaethylene glycol mono-n-dodecyl ether (C12E8). Compared to LPC, DM preserved specific activity of the solubilized Ca2+-ATPase during the chromatographic steps. Starting from 1/6 (3.8 mg) of the total amount of Ca2+-ATPase expressed in light membranes, 800 mug could be routinely purified to 50% purity by metal affinity chromatography and then 200 mug to 70% with Reactive red chromatography. The purified Ca2+-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 mumol ATP hydrolyzed/min/mg Ca2+-ATPase. It was stable and active for several days at 4degreesC or after removal of DM with Bio-beads and storage at -80degreesC. (C) 2002 Elsevier Science B.V. All rights reserved.