Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

Abstract

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.

Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli

“…hLTB 4 R). This result was particularly surprising, since optimizing the codon usage in the hLTB 4 R gene has been reported to dramatically increase expression levels in E. coli [18,26]. This result suggests that codon optimization may be more effective than supplementing rare tRNAs when expressing human genes in E. coli.…”

“…However, expression of the hLTB 4 receptor could not be detected in any of the cell strains, either by Coomassie-stained SDS-PAGE or Western blot (data not shown).…”

“…The hNK 2 R and hLTB 4 R genes were subcloned into the pET23b vector using NdeI and NotI; using the NdeI site eliminated the T7 tag. The genes were placed in frame to allow the encoding of a C-terminal hexa-histidine tag to facilitate purification.…”

“…One is to produce the membrane protein in its functional form; this approach usually results in low-level expression [4]. The second is to express the membrane protein as inclusion bodies; these must then be solubilized and then refolded to produce the native, functional form of the protein.…”

“…For only two receptors, human NK 1 receptor (hNK 1 R) and mouse NK 1 receptor (mNK 1 R) were we able to expressed large quantities of inclusion bodies. In no case, even for hBLT 4 receptor, was expression of the GPCR genes improved by the use of E. coli Rosetta™ strains, which are designed to compensate for differences in codon usage by the expression of rare tRNAs.…”