Summary
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.
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The two authors contributed equally to this work.
This project was partially supported by a grant from Army Medical Research Program of China (No. 08G168).
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Wang, C., Wang, Y., Wang, M. et al. Signal peptide and denaturing temperature are critical factors for efficient mammalian expression and immunoblotting of cannabinoid receptors. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 32, 299–302 (2012). https://doi.org/10.1007/s11596-012-0052-4
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DOI: https://doi.org/10.1007/s11596-012-0052-4