Wang Chenyun, Wang Yingying, Wang Miao, Chen Jiankui, Yu Nong, Song Shiping, Kaminski Norbert E, Zhang Wei
Department of Pharmaceutical Administration, PLA General Hospital, Beijing, 100850, China.
Outpatient Department of Xiyuan, PLA General Hospital, Beijing, 100850, China.
J Huazhong Univ Sci Technolog Med Sci. 2012 Apr;32(2):299-302. doi: 10.1007/s11596-012-0052-4. Epub 2012 Apr 20.
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 immuno-blotting. 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 immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.
许多研究人员采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看不到直接证明有效蛋白表达的免疫印迹数据。在本研究中，我们证明了常规的哺乳动物表达系统无法正确表达大麻素受体蛋白。通过在大麻素受体肽前赋予一个外源性信号肽，这种低效表达得到了挽救。此外，发现人工合成的大麻素受体在常规样品变性温度（即≥95°C）下会聚集，在免疫印迹分析时形成一条高分子量条带。只有变性温度≤75°C时，才会在预测分子量处产生清晰条带。总体而言，我们表明大麻素受体的高效哺乳动物表达需要一个信号肽序列，并描述了免疫印迹分析中对低样品变性温度的要求。这些发现为膜受体的高效哺乳动物表达和免疫印迹提供了非常有用的信息。
