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 hete rologous sarcoplasmic reticulum Ca2+-ATPase (Serca1a), Serca1a was tagged w ith 6 histidines at its C-terminal end and overexpressed using the baculovi rus-Sf9 system. In a first trial, Serca1a accounted for 24% of membrane pro teins, 95% of which were inactive. Glucose in the culture medium reduced th e production of Serca1a to 3 to 5% of membrane proteins and all Serca1a was active. Seventy-five percent of active Serca1a was solubilized by C12E8 in the presence of phosphatidylcholine under conditions avoiding denaturation . Purification by Ni2+-nitrilotriacetic acid affinity chromatography was tr ied, 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 2 5% and Serca1a was stable for at least 1 week at 0 degreesC, 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 Ca2+ a ffinity (2 muM at pH 7). (C) 2001 Academic Press.