Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors

Many researchers employed mammalian expression system to artificially express can-nabinoid 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...

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Published inJournal of Huazhong University of Science and Technology. Medical sciences Vol. 32; no. 2; pp. 299 - 302
Main Author 王辰允 王颖莹 王淼 陈建奎 于农 宋世平 Norbert E.KAMINSKI 张伟
Format Journal Article
LanguageEnglish
Published Heidelberg Huazhong University of Science and Technology 01.04.2012
Department of Pharmaceutical Administration,Beijing 100850,China%Outpatient Department of Xiyuan,PLA General Hospital,Beijing 100850,China%Department of Clinical Laboratory,No. 307 Hospital,Academy of Military Medical Sciences,Beijing 100071,China%Department of Pharmacology and Toxicology and the Center for Integrative Toxicology,Michigan State University,East Lansing,M148824,USA
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ISSN1672-0733
1993-1352
DOI10.1007/s11596-012-0052-4

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Summary:Many researchers employed mammalian expression system to artificially express can-nabinoid 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 ex-pression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor pep-tide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under rou-tine 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 pre-dicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabi-noid receptors need a signal peptide sequence, and described the requirement for a low sample dena-turing temperature in immuno-blot analysis. These findings provide very useful information for effi-cient mammalian expression and immuno-blotting of membrane receptors.
Bibliography:42-1679/R
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The two authors contributed equally to this work.
ISSN:1672-0733
1993-1352
DOI:10.1007/s11596-012-0052-4