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
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.