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

Paul K. Goldsmith, Gao Feng Fan, Kausik Ray, Joseph Shiloach, Peter McPhie, Kimberly V. Rogers, Allen M. Spiegel

Research output: Contribution to journalArticle

56 Citations (Scopus)

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 ~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.

Original languageEnglish (US)
Pages (from-to)11303-11309
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number16
DOIs
StatePublished - Apr 16 1999
Externally publishedYes

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Calcium-Sensing Receptors
Purification
Carbohydrate Sequence
Proteolysis
Proteins
Isoleucine
Nonsense Codon
HEK293 Cells
Gel permeation chromatography
G-Protein-Coupled Receptors
Electrophoresis
Lectins
Disulfides
Dimers
Gel Chromatography
Anions
Culture Media
Polyacrylamide Gel Electrophoresis
Complementary DNA
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression, purification, and biochemical characterization of the amino- terminal extracellular domain of the human calcium receptor. / Goldsmith, Paul K.; Fan, Gao Feng; Ray, Kausik; Shiloach, Joseph; McPhie, Peter; Rogers, Kimberly V.; Spiegel, Allen M.

In: Journal of Biological Chemistry, Vol. 274, No. 16, 16.04.1999, p. 11303-11309.

Research output: Contribution to journalArticle

Goldsmith, Paul K. ; Fan, Gao Feng ; Ray, Kausik ; Shiloach, Joseph ; McPhie, Peter ; Rogers, Kimberly V. ; Spiegel, Allen M. / Expression, purification, and biochemical characterization of the amino- terminal extracellular domain of the human calcium receptor. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 16. pp. 11303-11309.
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