Purification of heterologous sarcoplasmic reticulum Ca2+-ATPase Serca1a allowing phosphoenzyme and Ca2+-affinity measurements.
We describe here a protocol to prepare milligrams of active and stable heterologous sarcoplasmic reticulum Ca(2+)-ATPase (Serca1a). Serca1a was tagged with 6 histidines at its C-terminal end and overexpressed using the baculovirus-Sf9 system. In a first trial, Serca1a accounted for 24% of membrane proteins, 95% of which were inactive. Glucose in the culture medium reduced the production of Serca1a to 3 to 5% of membrane proteins and all Serca1a was active. Seventy-five percent of active Serca1a was solubilized by C(12)E(8) in the presence of phosphatidylcholine under conditions avoiding denaturation. Purification by Ni(2+)-nitrilo-triacetic acid affinity chromatography was tried, but only 3% of active Serca1a remained bound to the column, as if the His-tag were not accessible. Yields of 43% were reached by purification on reactive red 120 columns when eluting with 2 M NaCl. The purity was about 25% and Serca1a was stable for at least 1 week at 0 degrees C. Typically, 500 ml of culture medium produced 3 mg of active Serca1a and 1 mg of purified active Serca1a allowing measurements of phosphoenzyme (2 nmol/mg) or Ca(2+) affinity (2 microM at pH 7).
Miras
R
R
Commissariat à l'Energie Atomique, Département de Biologie Moléculaire et Structurale, Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche CEA-CNRS-UJF 5090, 17 rue des Martyrs, Grenoble Cedex 09, 38054, France.
Cuillel
M
M
Catty
P
P
Guillain
F
F
Mintz
E
E
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012995:Solubility; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics
D002413:Cations, Divalent; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium