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.
Lenoir
Guillaume
G
CEA, Centre d'Etudes de Saclay, Département de Biologie Cellulaire et Moléculaire, Section de Biophysique des Protéines Membranaires, Unité de Recherche Associée 2096 of the CNRS, Bât. 528, 91191 Cedex, Gif-sur-Yvette, France.
Menguy
Thierry
T
Corre
Fabienne
F
Montigny
Cédric
C
Pedersen
Per A
PA
Thinès
Denyse
D
le Maire
Marc
M
Falson
Pierre
P
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D008854:Microscopy, Electron; D009571:Nitrilotriacetic Acid; D010957:Plasmids; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure; D013696:Temperature; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014227:Triazines
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014227:Triazines; C003213:Procion Brilliant Red M-2BS; D000252:Calcium-Transporting ATPases; D009571:Nitrilotriacetic Acid