tailieunhanh - Báo cáo khoa học: Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor

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 histamineH1 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). | Eur. J. Biochem. 271 2636-2646 2004 FEBS 2004 doi Large-scale overproduction functional purification and ligand affinities of the His-tagged human histamine H1 receptor Venkata R. P. Ratnala1 Herman G. P. Swarts2 Jenny VanOostrum2 Rob Leurs3 Huub J. M. DeGroot1 Remko A. Bakker3 and Willem J. DeGrip1 2 1 Leiden Institute of Chemistry Leiden University department of Biochemistry UMC-160 Nijmegen Center for Molecular Life Sciences University Medical Center Nijmegen 3Department of Pharmacochemistry Division of Medicinal Chemistry Leiden Amsterdam Center for Drug Research Vrije Universiteit Amsterdam the Netherlands 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-SÍ9 . As judged from its ligand affinity profile functional receptor could be expressed at high levels 30-40 pmol per 106 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 chro matography 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 .