Present address: Laboratory of Molecular Biology, NIDDK, NIH, Building 50/4503, 50 South Drive, Bethesda, MD 20892-8030, USA.
Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli
Article first published online: 30 OCT 2003
European Journal of Biochemistry
Volume 269, Issue 1, pages 82–92, January 2002
How to Cite
Weiß, H. M. and Grisshammer, R. (2002), Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli. European Journal of Biochemistry, 269: 82–92. doi: 10.1046/j.0014-2956.2002.02618.x
- Issue published online: 30 OCT 2003
- Article first published online: 30 OCT 2003
- (Received 6 August 2001, accepted 22 October 2001)
- adenosine A2a receptor;
- G-protein-coupled receptor;
- maltose-binding protein fusion;
- functional solubilization
The adenosine A2a 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 A2a 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 [3H]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 [3H]ZM241385 with a Kd of 0.19 nm and an average Bmax of 13.7 nmol·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.