Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase.
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 degrees C to 18 degrees 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-alpha-lysophosphatidylcholine (LPC), 50% with n-dodecyl beta-D-maltoside (DM) and 25% with octaethylene glycol mono-n-dodecyl ether (C(12)E(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 microg could be routinely purified to 50% purity by metal affinity chromatography and then 200 microg 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 micromol ATP hydrolyzed/min/mg Ca(2+)-ATPase. It was stable and active for several days at 4 degrees C or after removal of DM with Bio-beads and storage at -80 degrees C.[1]References
- Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. Lenoir, G., Menguy, T., Corre, F., Montigny, C., Pedersen, P.A., Thinès, D., le Maire, M., Falson, P. Biochim. Biophys. Acta (2002) [Pubmed]
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