NDLI: Design, generation, and testing of mammalian expression modules that tag membrane proteins.

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Design, generation, and testing of mammalian expression modules that tag membrane proteins.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Pang, John Zeng, Xiaokun Xiao, Rui-ping Lakatta, Edward G. Lin, Li
Description	Country affiliation: United States Author Affiliation: Pang J ( Laboratory of Cardiovascular Sciences, National Institute on Aging, National Institute of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.)
Abstract	The expression of mammalian membrane proteins in laboratory cell lines allows their biological functions to be characterized and carefully dissected. However, it is often difficult to design and generate effective antibodies for membrane proteins in the desired studies. As a result, expressed membrane proteins cannot be detected or characterized via common biochemical approaches such as western blotting, immunoprecipitation, or immunohistochemical analysis, and their cellular behaviors cannot be sufficiently investigated. To circumvent such roadblocks, we designed and generated two sets of expression modules that consist of sequences encoding for three essential components: (1) a signal peptide from human receptor for advanced glycation end products that targets the intended protein to the endoplasmic reticulum for cell surface expression; (2) an antigenic epitope tag that elicits specific antibody recognition; and (3) a series of restriction sites that facilitate subcloning of the target membrane protein. The modules were designed with the flexibility to change the epitope tag to suit the specific tagging needs. The modules were subcloned into expression vectors, and were successfully tested with both Type I and Type III human membrane proteins: the receptor for advanced glycation end products, the Toll-like receptor 4, and the angiotensin II receptor 1. These expressed membrane proteins are readily detected by western blotting, and are immunoprecipitated by antibodies to their relative epitope tags. Immunohistochemical and biochemical analyses also show that the expressed proteins are located at cell surface, and maintain their modifications and biological functions. Thus, the designed modules serve as an effective tool that facilitates biochemical studies of membrane proteins.
ISSN	09618368
e-ISSN	1469896X
DOI	10.1002/pro.136
Journal	Protein Science
Issue Number	6
Volume Number	18
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	2009-06-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Membrane Proteins Biosynthesis Chemistry Protein Biosynthesis Advanced Glycosylation End Product-specific Receptor Metabolism Cell Line Cell Membrane Epitopes Genetic Vectors Receptor, Angiotensin, Type 1 Receptors, Immunologic Recombinant Fusion Proteins Toll-like Receptor 4 Transfection Research Support, N.i.h., Extramural Research Support, N.i.h., Intramural Research Support, Non-u.s. Gov't Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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Design, generation, and testing of mammalian expression modules that tag membrane proteins.

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