Expression, Purification, and Biochemical Characterization of the Amino-terminal Extracellular Domain of the Human Calcium Receptor

• Published in: The Journal of biological chemistry Vol. 274; no. 16; pp. 11303 - 11309
• Main Authors: Goldsmith, P K, Fan, G F, Ray, K, Shiloach, J, McPhie, P, Rogers, K V, Spiegel, A M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 16.04.1999
• Subjects: Amino Acid Sequence; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - isolation & purification; Calcium-Binding Proteins - metabolism; Cell Line; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Circular Dichroism; Dimerization; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Sequence Data; Protein Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.16.11303

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 ∼78-kDa protein on SDS-polyacrylamide gel electrophoresis and was found to be a disulfide-linked dimer. Its NH 2 -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 Ca 2+ activation of this G protein-coupled receptor.