Signal peptide and denaturing temperature are critical factors for efficient mammalian expression and immunoblotting of cannabinoid receptors.
Signal peptide and denaturing temperature are critical factors for efficient mammalian expression and immunoblotting of cannabinoid receptors.
复制标题
DOI：
10.1007/s11596-012-0052-4
复制
发表时间：
2012-04
影响因子：
--
通讯作者：
Zhang, Wei
中科院分区：
文献类型：
--
作者：
Wang, Chenyun;Wang, Yingying;Wang, Miao;Chen, Jiankui;Yu, Nong;Song, Shiping;Kaminski, Norbert E.;Zhang, Wei
关键词：
许多研究人员利用哺乳动物表达系统来人工表达大麻素受体，但在相关研究报告中几乎找不到直接证明有效蛋白表达的免疫印迹数据。在本研究中，我们证明大麻素受体蛋白无法用常规哺乳动物表达系统正确表达。通过在大麻素受体肽之前赋予外源信号肽，可以挽救这种低效表达。此外，通过免疫印迹分析，发现人工合成的大麻素受体在常规样品变性温度（即≥95°C）下聚集，形成大分子量条带。只有变性温度≤75°C 才能在预测的分子量处产生清晰的条带。总的来说，我们表明哺乳动物大麻素受体的有效表达需要信号肽序列，并描述了免疫印迹分析中低样品变性温度的要求。这些发现为膜受体的有效哺乳动物表达和免疫印迹提供了非常有用的信息。
Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors.
登录
查看更多内容
影响因子：
3.4
作者：
Hoffman, AF;Macgill, AM;Lupica, CR
通讯作者：
Lupica, CR
影响因子：
64.8
作者：
MUNRO, S;THOMAS, KL;ABUSHAAR, M
通讯作者：
ABUSHAAR, M
影响因子：
4.4
作者：
Samson, MT;Small-Howard, A;Turner, H
通讯作者：
Turner, H
影响因子：
1.6
作者：
Yeliseev, Alexel;Zoubak, Lioudmila;Gawrisch, Klaus
通讯作者：
Gawrisch, Klaus
影响因子：
1.6
作者：
Zhang, Rundong;Kim, Tae-Kang;Song, Zhao-Hui
通讯作者：
Song, Zhao-Hui