Manifestation of milligram quantities of functional stable G protein-coupled receptors (GPCR)

Manifestation of milligram quantities of functional stable G protein-coupled receptors (GPCR) for high-resolution structural studies remains a challenging task. partners. Manifestation Schisanhenol by induction with either IPTG (in BL21(DE3) cell ethnicities) or by auto-induction (in KRX cells) were compared. While the N-terminal location of the HaloTag resulted in high levels of manifestation of the fusion CB2 the recombinant receptor was not practical. However when the HaloTag was placed in the C-terminal location a fully active receptor was produced irrespective of induction method or bacterial strain used. For purification the fusion protein was captured onto HaloLink resin in the presence of detergents. Treatment with specific TEV protease released the CB2 upon washing. CD95 To our knowledge this study represents the first example of expression surface immobilization and purification of a functional GPCR Schisanhenol using HaloTag technology. Introduction The cannabinoid receptors CB1 and CB2 belong to the large family of G protein-coupled receptors (GPCRs) and are central to the endocannabinoid system that also includes endocannabinoid ligands as well as enzymes of their respective anabolic and catabolic pathways. CB2 is usually predominantly Schisanhenol expressed by cells of the immune system and is a primary target for the treatment of immune disorders inflammatory diseases and pain sensing [1-4]. The primary signaling function of CB2 appears to be the inhibition of cAMP accumulation [5]. Rational design of specific drugs for controlling function of CB2 relies on functional and structural information around the receptor that requires highly pure preparations of correctly folded receptor. Previously we reported on expression of CB2 in membranes as a fusion with maltose binding protein (MBP) thioredoxin (TrxA) and two small affinity tags a Strep-tag III and a polyhistidine tag [6] [7]. Purification via the affinity tags placed at opposing ends of the receptor and cleavage of the fusion by tobacco etch computer virus protease yielded up to 0.4 mg of active CB2 per liter of culture [7]. However in the presence of detergents required for solubilization of CB2 the affinity of the Strep-tag for the StrepTactin resin is usually relatively low resulting in substantial losses during this chromatographic step. Hence to achieve a higher yield and purity of the receptor optimization of purification conditions is usually desired. In order to improve the yield of CB2 in the equilibrium-based chromatographic step we take advantage of the HaloTag (Promega) a 34 kDa catalytically inactive derivative of Haloalkane dehalogenase from that rapidly specifically and covalently binds to synthetic chloroalkane ligands. The HaloTag technology [8 9 relies on the binding of the Halo-tagged target protein to chloroalkane linker attached to the chromatographic resin. Since the binding of the HaloTag to the resin is usually virtually irreversible an extensive washing procedure can be employed which enhances the purity and yield of the target protein substantially. Thus a Halo-tagged protein can be covalently immobilized on a resin efficiently purified and eluted from your resin upon cleaving of the fusion with TEV protease at a specific site located between the target protein and the tag. Recombinant expression of CB2 in bacterial (expression system the CB2 produced in yeast was reported to be non-homogeneously glycosylated and non-functional [10]. One of the major troubles of expressing mammalian proteins in cells is related to the fact that Schisanhenol heterologous protein expression often results in low expression levels and/or poor solubility of the produced protein. These problems are frequently overcome by introducing N-terminal expression tags (i.e MBP or HaloTag). Integral membrane proteins present a particular challenge since their expression in often results in formation of inclusion bodies that require subsequent refolding. This can be avoided by targeting these membrane proteins for Schisanhenol insertion into the plasma membrane of by adding a tag such as MBP. This approach has been shown to yield properly folded and functional membrane proteins [11 12 Production of Schisanhenol recombinant proteins in by induction with IPTG is usually a widely used methodology. Typically in a host cell such as BL21(DE3) the target protein is usually encoded in a plasmid under control of the T7 promoter that is recognized by T7.