Heitz F, Nay C, Guenet C
Marion Merrell Research Institute, Strasbourg, France.
J Recept Signal Transduct Res. 1997 Jan-May;17(1-3):305-17. doi: 10.3109/10799899709036611.
Human M2 receptors were expressed using the baculovirus expression system in three different insect cell lines: Sf9, Sf21 and High5. The level of expression was slightly increased in Sf21 cells versus Sf9 cells. In contrast, High5 cells were not able to produce more recombinant protein than Sf9. We also show that in both Spodoptera frugiperda cell lines a peak of expression was reached after 6 days of infection, whereas in High5 cells, the maximum of expression occurred after 3 days. Immunodetection of m2 muscarinic receptor clearly shows that the expressed protein undergoes significant proteolysis in both the Sf9 and High5 cells, whereas in the Sf21 cells this phenomenon was less detectable. Additionally, we show that in all three cell lines, the expressed recombinant receptor was functional in that it was able to stimulate GTP gamma S binding in the presence of exogenous G-proteins. Analysis of the population of G-proteins (G alpha i, G alpha o and G beta common) in Sf21 and High5 cells is provided.
使用杆状病毒表达系统在三种不同的昆虫细胞系（Sf9、Sf21和High5）中表达人M2受体。与Sf9细胞相比，Sf21细胞中的表达水平略有增加。相比之下，High5细胞产生的重组蛋白并不比Sf9细胞多。我们还表明，在两种草地贪夜蛾细胞系中，感染6天后达到表达峰值，而在High5细胞中，最大表达量在3天后出现。m2毒蕈碱受体的免疫检测清楚地表明，在Sf9和High5细胞中，表达的蛋白都经历了显著的蛋白水解，而在Sf21细胞中，这种现象不太明显。此外，我们表明，在所有三种细胞系中，表达的重组受体都具有功能，因为它能够在外源G蛋白存在的情况下刺激GTPγS结合。文中还提供了Sf21和High5细胞中G蛋白（Gαi、Gαo和Gβ共同亚基）群体的分析。
