High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line.
The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.
Camponova
Paméla
P
Centre de Biochimie Structurale, UMR CNRS 5048, INSERM 554, 29 rue de Navacelles, 34090 Montpellier Cedex, France.
Baud
Stéphanie
S
Mattras
Hélène
H
Duroux-Richard
Isabelle
I
Bonnafous
Jean-Claude
JC
Marie
Jacky
J
D000595:Amino Acid Sequence; D001345:Autoradiography; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D043782:Receptor, Bradykinin B2 / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*
D008024:Ligands; D043782:Receptor, Bradykinin B2; D013752:Tetracycline