Opefi, Chikwado A and Tranter, Dale and Smith, Steven O and Reeves, Philip J (2015) 'Construction of Stable Mammalian Cell Lines for Inducible Expression of G Protein-Coupled Receptors.' MEMBRANE PROTEINS - PRODUCTION AND FUNCTIONAL CHARACTERIZATION, 556. pp. 283-305. ISSN 0076-6879

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Abstract

The large-scale expression of many membrane proteins, including the members of the G protein-coupled receptor superfamily, in a correctly folded and fully functional form remains a formidable challenge. In this chapter, we focus on the construction of stable mammalian cell lines to overcome this hurdle. First, we will outline the steps for establishing a tightly regulated gene expression system in human HEK293S cells. This system utilizes separate plasmids containing components of well-defined genetic control elements from the Escherichia coli tetracycline operon to control the powerful cytomegalovirus immediate early enhancer/promoter. Next, we describe the assembly of this expression system into HEK293S cells and a derivative cell line devoid of complex N-glycosylation. Finally, we describe methods for the growth of these cells lines in scalable suspension culture for the preparation of milligram amounts of recombinant protein.

Item Type:	Article
Uncontrolled Keywords:	Animals; Humans; Escherichia coli; Tetracycline; Receptors, G-Protein-Coupled; Recombinant Proteins; Cell Culture Techniques; Cloning, Molecular; Gene Expression Regulation; Glycosylation; Operon; HEK293 Cells
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Faculty of Science and Health Faculty of Science and Health > Life Sciences, School of
SWORD Depositor:	Elements
Depositing User:	Elements
Date Deposited:	16 Apr 2015 15:40
Last Modified:	15 Jan 2022 00:30
URI:	http://repository.essex.ac.uk/id/eprint/13574

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