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Methods Enzymol. 2015;556:283-305. doi: 10.1016/bs.mie.2014.12.020. Epub 2015 Mar 20.

Construction of stable mammalian cell lines for inducible expression of G protein-coupled receptors.

Author information

1
Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom.
2
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
3
Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom. Electronic address: preeves@essex.ac.uk.

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.

KEYWORDS:

Crystallization; GnTI(−); HEK293S; Immunoaffinity purification; N-glycosylation; NMR; Rhodopsin; Sodium butyrate; Tetracycline inducible

PMID:
25857787
DOI:
10.1016/bs.mie.2014.12.020
[Indexed for MEDLINE]

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