title: Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor.
abstract: We purified the extracellular domain (ECD) of the human calcium receptor (hCaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA containing an isoleucine 599 nonsense mutation. A combination of lectin, anion exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purified ECD ran as an approximately 78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal sequence, carbohydrate content, and CD spectrum were defined. Tryptic proteolysis studies showed two major sites accessible to cleavage. These studies provide new insights into the structure of the hCaR ECD. Availability of purified ECD protein should permit further structural studies to help define the mechanism of Ca2+ activation of this G protein-coupled receptor.
authors: Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: P K
authors: PK
authors: Goldsmith
authors: G F
authors: GF
authors: Fan
authors: K
authors: K
authors: Ray
authors: J
authors: J
authors: Shiloach
authors: P
authors: P
authors: McPhie
authors: K V
authors: KV
authors: Rogers
authors: A M
authors: AM
authors: Spiegel
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D018076:DNA, Complementary; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D002135:Calcium-Binding Proteins; D018076:DNA, Complementary