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.
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