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

Abstract

In combining fluorescence measurements with ligand binding assays, the versatility of the EGFP C-terminally fused to the human mu opioid receptor (EGFP-hMOR) has been exploited to notably improve the expression level of functional G protein-coupled receptors in Drosophila S2 cells. A selected array of efficient optimization approaches is presented herein, ranging from a cell-sorting method, allowing for a substantial enrichment in EGFP-hMOR expressing cells, to the addition of chemical and pharmacological chaperones, significantly enhancing the yield and the activity of the expressed receptors. Consistent with previous studies, significant discrepancies were observed between the total amounts of fluorescent receptors over a limited subpopulation capable of ligand binding, even after expression optimization. Subsequently, membrane isopycnic centrifugation experiments allowed to separate the ligand binding active from the non-active membrane fraction, the latter most probably containing misfolded receptors. Taken together, these results illustrate a coherent set of advantageous productive and preparative methods for the production of GPCRs in the highly valuable Drosophila S2 expression system.

Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

“…In addition, DES has been reported to have advantageous bioprocess characteristics, such as high cell density attained, low cost culture medium needed and continuous bioprocess possibility (Affleck and Walker, 2008;Batista et al, 2008Batista et al, , 2009Bovo et al, 2008;Brillet et al, 2008;Galesi et al, 2008;Iwaki and Castellino, 2008;Jorge et al, 2008;Park et al, 2008;Santos et al, 2007;Swiech et al, 2008aSwiech et al, , 2008b. In previous publications Yokomizo et al, 2007) we have shown that the DES indeed represents a novel and promising approach for the production of the rabies virus glycoprotein (RVGP).…”

Monitoring the human β1, β2, β3 adrenergic receptors expression and purification in Pichia pastoris using the fluorescence properties of the enhanced green fluorescent protein

“…Fermentation of Pichia pastoris cells has been optimized over years and it is possible routinely to obtain 100 g/l of dry cell weight whereas 5 g/l are obtained in flasks. The functionality of a recombinant GPCR is an important issue as recently discussed (Brillet et al 2008). In Pichia pastoris, the three beta-adrenergic receptor subtypes in fusion with EGFP were not able to bind specific ligands (data not shown) as for the human mu-opioid receptor fused to EGFP (Sarramegna et al 2005).…”

Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression

“…The simplest method involves the repeated treatment of a polyclonal population with high concentrations of the selection antibiotic in order to enrich for a high-producer subpopulation [ 6 ]. Cell lines expressing surface proteins can also be isolated using immuno-magnetic selection [ 5 , 45 , 46 ]. However, in both cases, the corresponding subpopulations remain polyclonal and are therefore still genetically inhomogeneous, possibly reducing the stability of protein expression over time due to differences in growth kinetics.…”