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目的评估不同转染及培养条件下COS1细胞过表达钙离子ATP酶SERCA1a的量及活性,探讨影响真核细胞目标蛋白质表达量及活性的因素。方法应用常规的全量或者半量DNA、lipofactamin和plus reagent转染COS1细胞,37℃、34℃或31℃培养2至5d。提取微粒体蛋白后定量并测定SERCA1a的ATP酶活性。结果微粒体蛋白量在细胞培养3至4d达到高峰,改变转染及细胞培养条件,未见明显变化;降低细胞培养温度及提高DNA转染用量,可增加SERCA1a表达量;SERCA1a表达量在8μg DNA转染细胞31℃培养3d达到最高,然而降低细胞培养温度后,SERCA1a的ATP酶活性及EP生成量也随之下降;31℃表达的SERCA1a,其ATP酶活性降低比EP生成量减少的幅度更大,并且ATP酶活性及EP生成量随着细胞培养时间延长而增加。结论应用常规的半量转染试剂、全量DNA瞬时转染COS1细胞,37℃培养3-4d可获得最大量具有正常酶活性的SERCA1a蛋白。降低培养温度虽然可以提高外源性SERCA1a的表达,但却可能影响细胞内蛋白质的准确折叠及正常降解。
Objective To evaluate the quantity and activity of SERCA1a overexpressing calcium ion ATPase in COS1 cells under different transfection and culture conditions and to explore the factors influencing the target protein expression and activity of eukaryotic cells. METHODS: COS1 cells were transfected with normal or semi-quantitative DNA, lipofactamin and plus reagents and cultured at 37 ℃, 34 ℃ or 31 ℃ for 2 to 5 days. The microsomal protein was extracted and quantitated and assayed for ATPase activity of SERCA1a. Results The amount of microsomal protein peaked at 3 to 4 days and the transfection and cell culture conditions were changed. No significant changes were observed. The cell culture temperature and the amount of DNA transfection could increase the expression of SERCA1a. The expression of SERCA1a in 8μg DNA The transfected cells had the highest level at 31 ℃ for 3 days, however, the decrease of ATPase activity and EP production of SERCA1a decreased after the cell culture temperature was decreased. SERCA1a expressed at 31 ℃ reduced the activity of ATPase more than that of EP Large, and ATPase activity and EP production increased with cell culture time. CONCLUSIONS: Transfection of COS1 cells with total amount of DNA using conventional semi-transfection reagents can maximize the amount of SERCA1a protein with normal enzymatic activity at 37 ℃ for 3-4 days. Decreasing the culture temperature may improve the exogenous SERCA1a expression but may affect the accurate folding and normal degradation of intracellular proteins.