title: Signal peptide and denaturing temperature are critical factors for efficient mammalian expression and immunoblotting of cannabinoid receptors.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Administration, PLA General Hospital, Beijing, 100850, China.
authors.forename: Chenyun
authors.initials: C
authors.lastname: Wang
authors.affiliation: Outpatient Department of Xiyuan, PLA General Hospital, Beijing, 100850, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Clinical Laboratory, No. 307 Hospital, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Clinical Laboratory, No. 307 Hospital, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Jiankui
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory, No. 307 Hospital, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Nong
authors.initials: N
authors.lastname: Yu
authors.affiliation: Department of Clinical Laboratory, No. 307 Hospital, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Shiping
authors.initials: S
authors.lastname: Song
authors.affiliation: Department of Pharmacology and Toxicology and the Center for Integrative Toxicology, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Norbert E
authors.initials: NE
authors.lastname: Kaminski
authors.affiliation: Department of Clinical Laboratory, No. 307 Hospital, Academy of Military Medical Sciences, Beijing, 100071, China. zhangwei@yahoo.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D011489:Protein Denaturation; D021382:Protein Sorting Signals; D043882:Receptors, Cannabinoid / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D021382:Protein Sorting Signals; D043882:Receptors, Cannabinoid