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 in | Journal of Huazhong University of Science and Technology. Medical sciences Vol. 32; no. 2; pp. 299 - 302 |
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Main Author | |
Format | Journal Article |
Language | English |
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 |
Subjects | |
Online Access | Get full text |
ISSN | 1672-0733 1993-1352 |
DOI | 10.1007/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. |
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Bibliography: | 42-1679/R ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 The two authors contributed equally to this work. |
ISSN: | 1672-0733 1993-1352 |
DOI: | 10.1007/s11596-012-0052-4 |