title: Construction of stable mammalian cell lines for inducible expression of G protein-coupled receptors.
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.
authors: Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom.
authors: Chikwado A
authors: CA
authors: Opefi
authors: Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom.
authors: Dale
authors: D
authors: Tranter
authors: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors: Steven O
authors: SO
authors: Smith
authors: Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom. Electronic address: preeves@essex.ac.uk.
authors: Philip J
authors: PJ
authors: Reeves
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006031:Glycosylation; D057809:HEK293 Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D009876:Operon; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013752:Tetracycline / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D013752:Tetracycline
keywords: Crystallization; GnTI(−); HEK293S; Immunoaffinity purification; N-glycosylation; NMR; Rhodopsin; Sodium butyrate; Tetracycline inducible