Construction of Recombinant Cell Lines for GPCR Expression.

Abstract

Large-scale recombinant expression of G protein-coupled receptors (GPCRs) is required for structure and function studies where there is a need for milligram amounts of protein in pure form. Here we describe a procedure for the construction of human embryonic kidney 293S (HEK293S) stable cell lines for inducible expression of the gene encoding bovine rhodopsin. The HEK293S cell line is particularly suitable for this application because of several favorable properties as a recombinant host including: its ease of transfection, its capacity for handling large amounts of protein cargo, and its ability to perform the necessary co- and post-translational modifications required for correct folding and processing of complex membrane proteins such as GPCRs. The procedures described here will focus on the HEK293S GnTI- cell line, an HEK293S derivative that is widely used for the production of glycoproteins modified homogeneously with truncated N-glycans.