Dimethylsulphoxide as a tool to increase functional expression of heterologously produced GPCRs in mammalian cells

Abstract

High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B 2 R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B 2 R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells.

The Structure of the Neuropeptide Bradykinin Bound to the Human G‐Protein Coupled Receptor Bradykinin B2 as Determined by Solid‐State NMR Spectroscopy

“…Large‐scale cultures (25 L) were grown using a Wave bioreactor (SYSTEM20/50, WAVE BIOTECH). Membrane preparation and [ 3 H] BK binding experiments were performed as described in references 33–35. K d values of 0.4±0.1 n M were obtained and compare favorably to those reported for human decidual cells (0.85 n M ) 50.…”

“…Preparation of human GPCR B 2 R: B 2 R was produced in Sf9 cells using the baculovirus system. Expression constructs and optimized production conditions were reported earlier 33–35. Large‐scale cultures (25 L) were grown using a Wave bioreactor (SYSTEM20/50, WAVE BIOTECH).…”

“…To date, a structural investigation of B 2 R‐bound BK was prevented by the lack of 1) B 2 R availability at a preparative scale and 2) a method for obtaining structural data of high‐affinity ligands bound to large membrane proteins. Recently, B 2 R could be overexpressed in mammalian cells and insect cells 33–35. Furthermore, solid‐state NMR spectroscopy has progressed, and data of high‐affinity ligands bound to large membrane proteins in a noncrystalline state have been reported 10.…”

Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells

“…Futhermore, a cooperative effect was observed when a combination of both compounds was added to the medium. When compared to similar studies conducted on several GPCRs with other expression systems, where it was shown that the main effect of these chaperones was to improve the functionality of the expressed receptors (André et al 2006;Shukla et al 2006;Sarramegna et al 2002), our results unexpectedly demonstrated that these molecules also increased the total amount of EGFP-hMOR. Whether this particular feature is related to the S2 cells system alone or is receptor dependent needs to be further investigated.…”

New Techniques to Express and Crystallize G Protein‐Coupled Receptors

“…Optimization of the culture conditions through the addition of additives, such as ligands [65,129] , amino acids [130] , and DMSO [130,131] , and by altering temperature [130] can increase expression by nearly 10 -fold [130] .…”