Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells.
Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.
Gan
Lu
L
Department of Physiology, Tufts University, School of Medicine, 136 Harrison Avenue, Boston, MA 02111-1800, USA.
Alexander
Joseph M
JM
Wittelsberger
Angela
A
Thomas
Beena
B
Rosenblatt
Michael
M
D019149:Bioreactors; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
C473449:PTH1R protein, human; D044168:Receptor, Parathyroid Hormone, Type 1; D011994:Recombinant Proteins