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Characterization of a Mouse Serotonin 5-HT3 Receptor Purified from Mammalian Cells
Authors:Ruud Hovius  Ana-Paula Tairi  Horst Blasey  Alain Bernard  † Kenneth Lundström  Horst Vogel
Institution:Laboratory of Physical Chemistry of Polymers and Membranes, Chemistry Department, Swiss Federal Institute of Technology, Lausanne;; Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., Geneva, Switzerland;and; Glaxo Wellcome, Medicines Research Centre, Stevenage, Hertfordshire, England
Abstract:Abstract: A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding ∼50% of the histidine-tagged 5-HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of ∼5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT3 receptor is a pentameric homooligomer. The secondary structure of the 5-HT3 receptor as determined by circular dichroism appeared to consist of mainly α-helices (50%) and β-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.
Keywords:Serotonin 5-HT3 receptor  Semliki Forest virus expression  Purification  Secondary structure  Circular dichroism  Ligand binding
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