Large‐scale overproduction, functional purification and ligand affinities of the His‐tagged human histamine H1 receptor

Abstract

This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera frugiperda-derived cell line (IPLB-Sf9). As judged from its ligand affinity profile, functional receptor could be expressed at high levels (30-40 pmol per 10 6 cells). Rapid proteolysis in the cell culture led to limited fragmentation, without loss of ligand binding, but could be efficiently suppressed by including the protease inhibitor leupeptin during cell culture and all subsequent manipulations. Effective solubilization of functional receptor with optimal recovery and stability required the use of dodecylmaltoside as a detergent in the presence of a high concentration of NaCl and of a suitable inverse agonist. Efficient purification of solubilized receptor could be achieved by affinity chromatography over nickel(II) nitrilotriacetic acid resin. Functional membrane reconstitution of purified H1 receptor was accomplished in mixed soybean lipids (asolectin). The final proteoliposomic H1 receptor preparation has a purity greater than 90% on a protein basis and displays a ligand binding affinity profile very similar to the untagged receptor expressed in COS-7 cells. In conclusion, we are able to produce pharmacologically viable H1 receptor in a stable membrane environment allowing economic large-batch operation. This opens the way to detailed studies of structure-function relationships of this medically and biologically important receptor protein by 3D-crystallography, FT-IR spectroscopy and solid-state NMR spectroscopy.

Delineation of Receptor‐Ligand Interactions at the Human Histamine H1 Receptor by a Combined Approach of Site‐Directed Mutagenesis and Computational Techniques – or – How to Bind the H1 Receptor

“…To fully understand the (agonist) ligand binding characteristics and the activation mechanism of GPCRs molecular modelers look forward to more structural information. For the human H 1 receptor, large-scale purification methods have recently resulted in milligram quantities of purified and functional receptor suitable for solid state NMR experiments [63]. Currently, studies are performed to determine the configuration of histamine bound to the receptor.…”

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

“…If we except harsh detergents such as SDS or N-lauroyl sarcosine (NLS), it is difficult to predict which detergent will be suitable for solubilization. Some methods such as immunoblot quantification [27,28] or fluorescence measurements using green fluorescent protein (GFP)-tagged receptors allow quantification of the total yield of solubilization [29][30][31][32]. In the latter experiments, two different GPCRs were expressed in the same host, Pichia pastoris.…”

“…Nevertheless, the hexa-Histagged m-opioid receptor solubilized in 0.1% SDS could be purified by IMAC with an excellent yield [30]. The introduction of a C terminus deca-His tag to the H 1 histamine receptor allowed, after solubilization in DM, the purification, in a single step, of the recombinant protein with a purification yield higher than 90% and an excellent recovery of function (up to 70%) [28]. In addition, demonstrating once again that each receptor has a specific behavior, Pawate et al [59] obtained only 14% purification yield and 36% recovery for the CHAPS-solubilized hexa-His-tagged thromboxane A2 receptor.…”

Solid‐state NMR for the analysis of high‐affinity ligand/receptor interactions

“…Recent successes expressing a voltage-gated channel (16) and rat aquaporin (17) in Pichia pastoris and calcium ATPase (18) from Saccharomyces cerevisiae offer new hope for their analyses by solid-state NMR. Although slightly more difficult to scale-up than yeast fermentation, protein expression in mammalian cells has also been used to provide the quantities of membrane receptor required for solid-state NMR and has been extensively exploited in the studies of the GPCRs rhodopsin (19) and the histamine H1 receptor (20,21).…”