The detergentsn-dodecyl -d-maltopyranoside (DDM) and fos-choline-12 (FC12) were purchased from Anatrace; SDS was purchased from Sigma and digitonin was purchased from Calbiochem

The detergentsn-dodecyl -d-maltopyranoside (DDM) and fos-choline-12 (FC12) were purchased from Anatrace; SDS was purchased from Sigma and digitonin was purchased from Calbiochem. proteins are indeed correctly folded, without requiring SKI-II radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional SKI-II membrane proteins for structural studies. Abbreviations:FSEC, fluorescence-detection size-exclusion chromatography; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; SEC, size-exclusion chromatography; 1AR, 1-adrenergic receptor; A1R, A1receptor; GFP, green fluorescent protein; PBS, phosphate-buffered saline Keywords:eukaryotic membrane proteins, overexpression, G protein-coupled receptors, transporters == Graphical abstract == == Highlights == Discrimination between active and inactive membrane proteins after overexpression is usually difficult. For three G protein-coupled receptors SKI-II tested in the baculovirus expression system, the vast majority of the protein expressed was inactive. Inducible stable mammalian cell lines are far more efficient at producing correctly folded membrane protein. A simple assay by Western blotting discriminates active from inactive material. == Introduction == Structure determination of integral membrane proteins requires the production of milligrammes of real, authentically folded protein for crystallisation[1]. As a natural prerequisite, the protein needs to be expressed in one of a number of heterologous expression systems, such asEscherichia coli, yeasts, insect cells or mammalian cells[2]. A number of expression strategies have been developed for each host system and many are now efficient for expression trials of hundreds of proteins in parallel[3]. A SKI-II popular strategy for the expression of membrane proteins inE. coliis to generate fusion proteins with SKI-II green fluorescent protein (GFP), which can be used as an Mouse monoclonal to FOXD3 indicator for both the quantity of protein expressed[4]and its relative stability upon detergent solubilisation by fluorescence-detection size-exclusion chromatography (FSEC)[5]. The power of this strategy is usually that fluorescence of the fusion protein expressed in bacteria discriminates between correctly folded membrane protein (the GFP tag is usually fluorescent) and misfolded, aggregated membrane protein (the GFP tag is not fluorescent)[6],[7]. InE. coli, it appears that the misfolded membrane protein promotes the formation of inclusion bodies and, once in an aggregate, the GFP is unable to fold and attain fluorescence. However, in eukaryotic cells, such as yeasts, insect cells used in the baculovirus expression system and in mammalian cells, GFP tagged to a membrane protein remains fluorescent regardless of whether the membrane protein is usually misfolded in the endoplasmic reticulum or correctly folded in the plasma membrane[8],[9],[10],[11]. Higher eukaryotes have an efficient quality control system in the endoplasmic reticulum so that only folded proteins exit the endoplasmic reticulum, whilst misfolded proteins are retained for degradation[12]. Thus, GFP is not an appropriate marker for the folding status of membrane proteins produced using either mammalian cells or the baculovirus expression system, although it is still useful in analysing the stability of a membrane protein in different detergents by FSEC. The baculovirus expression system has confirmed efficient for the production of many eukaryotic membrane proteins, such as G protein-coupled receptors (GPCRs)[13], some of which have been crystallised and their structures determined[14]. However, recombinant baculovirus is not a panacea and there are numerous proteins that are poorly expressed and there have also been reports that some membrane proteins are expressed predominantly in a misfolded state[2],[15]. This is a serious problem for structural biology, as it is not obvious from current methodology whether an overexpressed membrane protein is usually predominantly folded or misfolded. If misfolded material is usually inadvertently purified, then this will likely have a detrimental effect on the ability of the sample to crystallise and may also adversely affect the quality of any crystals obtained. The best way to determine whether misfolded material is present is usually.