Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor

Citation
Pk. Goldsmith et al., Expression, purification, and biochemical characterization of the amino-terminal extracellular domain of the human calcium receptor, J BIOL CHEM, 274(16), 1999, pp. 11303-11309
Citations number
25
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
274
Issue
16
Year of publication
1999
Pages
11303 - 11309
Database
ISI
SICI code
0021-9258(19990416)274:16<11303:EPABCO>2.0.ZU;2-L
Abstract
We purified the extracellular domain (ECD) of the human calcium receptor (h CaR) from the medium of HEK-293 cells stably transfected with a hCaR cDNA c ontaining an isoleucine 599 nonsense mutation. A combination of lectin, ani on exchange, and gel permeation chromatography yielded milligram quantities of >95% pure protein from 15 liters of starting culture medium. The purifi ed ECD ran as an similar to 78-kDa protein on SDS-polyacrylamide gel electr ophoresis and was found to be a disulfide-linked dimer. Its NH2-terminal se quence, carbohydrate content, and CD spectrum were defined. Tryptic proteol ysis studies showed two major sites accessible to cleavage. These studies p rovide new insights into the structure of the hCaR ECD. Availability of pur ified ECD protein should permit further structural studies to help define t he mechanism of Ca2+ activation of this G protein-coupled receptor.