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).
Copyright 2001 Academic Press.