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

Autor:	Opefi CA; Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom., Tranter D; Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom., Smith SO; Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA., Reeves PJ; Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom. Electronic address: preeves@essex.ac.uk.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2015; Vol. 556, pp. 283-305. Date of Electronic Publication: 2015 Mar 20.
DOI:	10.1016/bs.mie.2014.12.020
Abstrakt:	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. (© 2015 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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