转染及培养条件对外源性钙离子ATP酶SERCA1a表达的影响

The Effect of Conditions Applied for Transfection and Cell Culture on the Expression of Exogenous Calcium ATPase SERCA1a

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。

Obj ective The amount and activities of calcium ATPase SERCA1 a were evaluated to study the effect of different conditions on the expression of exogenous target proteins.Methods COS1 cells were transfected with routine amount or half amount of DNA,lipofectamine and plus reagent,and then cultured at 37 ℃,34 ℃ or 31 ℃ for 2 to 5 days.Microsomal proteins were prepared by ultracentrifuga-tion.SERCA1 a proteins were quantified and used for the determination of ATP hydrolysis and EP (auto-phosphorylated intermediate)formation.Results The amount of microsomal proteins reach the peak in cells cultured for 3 to 4 days and did not change with other conditions.SERCA1 a per mg microsomal pro-teins increased with more DNA transfection and lower temperature.Although ELISA showed the highest concentration of SERCA1a from 8μg DNA transfected cells cultured at 37 ℃ for 3 days,SERCA1a AT-Pase activity and EP formation were reduced whit the decrease in temperature.For SERCA1 a expressed at 3 1 ℃,ATPase activity decreased more strongly than EP formation did.Conclusion The optimal condition for exogenous SERCA1 a expression in transiently transfected COS1 cells should be half amount of trans-fection reagents,routine amount of DNA,and cell culture at 3 7 ℃ for 3 to 4 days.A decreased culture temperature can increase the expression of proteins in cells,but also increase protein misfolding and decrease' wrong'protein degradation.