Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli.
The adenosine A(2a) receptor belongs to the seven transmembrane helix G-protein-coupled receptor family, is abundant in striatum, vasculature and platelets and is involved in several physiological processes such as blood pressure regulation and protection of cells during anoxia. For structural and biophysical studies we have expressed the human adenosine A(2a) receptor (hA2aR) at high levels inserted into the Escherichia coli inner membrane, and established a purification scheme. Expression was in fusion with the periplasmic maltose-binding protein to levels of 10-20 nmol of receptor per L of culture, as detected with the specific antagonist ligand [(3)H]ZM241385. As the receptor C-terminus was proteolyzed upon solubilization, a protease-resistant but still functional receptor was created by truncation to Ala316. Addition of the sterol, cholesteryl hemisuccinate, allowed a stable preparation of functional hA2aR solubilized in dodecylmaltoside to be obtained, and, increased the stability of the receptor solubilized in other alkylmaltosides. Purification to homogeneity was achieved in three steps, including ligand affinity chromatography based on the antagonist xanthine amine congener. The purified hA2aR fusion protein bound [(3)H]ZM241385 with a K(d) of 0.19 nm and an average B(max) of 13.7 nmol x mg(-1) that suggests 100% functionality. Agonist affinities for the purified solubilized receptor were higher than those for the membrane-bound form. Sufficient pure, functional hA2aR can now be prepared regularly for structural studies.
Weiss
H Markus
HM
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK. weiss@aeskulab.de
Grisshammer
Reinhard
R
D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012995:Solubility; D014227:Triazines / Q000378:metabolism; D014230:Triazoles / Q000378:metabolism
D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; D014227:Triazines; D014230:Triazoles; C097270:ZM 241385