Expression of recombinant homo-oligomeric 5-hydroxytryptamine3 receptors provides new insights into their maturation and structure.
A recombinant baculovirus containing a mouse 5-hydroxytryptamine3 (5-HT3) receptor subunit cDNA under the control of the polyhedrin promoter was shown to direct the production of large amounts of functional 5-HT3 receptor in insect cells, as assayed by Western blotting and ligand binding. After solubilization, the receptor was purified to homogeneity by affinity chromatography and characterized pharmacologically. The ligand binding characteristics of the recombinant receptor were essentially identical to those of the native receptor, both before and after purification. Only fully glycosylated receptors bound to the ligand affinity resin, although subsequent removal of the sugar did not affect ligand binding. Visualization of the purified receptor using electron microscopy showed that the receptor preparation contained a homogeneous population of pentameric doughnut-shaped particles. The general appearance of the recombinant homo-oligomeric channels was indistinguishable from that of native 5-HT3 receptors. Yields of purified receptors were of the order of 200 micrograms/3 liters of original culture. The amount and homogeneity of the purified receptor are sufficient to begin preliminary crystallization trials.
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Centre for Protein Engineering, Medical Research Council Centre, Cambridge, United Kingdom.
Stauffer
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Lummis
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D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006031:Glycosylation; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008854:Microscopy, Electron; D011401:Promoter Regions, Genetic; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D046911:Macromolecular Substances; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins