TAILIEUCHUNG - Báo cáo Y học: Puri®cation and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli

The adenosine A2a receptor belongs to the seven trans-membrane 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 A2areceptor (hA2aR) at high levels inserted into the Escherichia coliinner membrane, and established a puri®cation scheme. | Eur. J. Biochem. 269 82-92 2002 FEBS 2002 Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli H. Markus WeiB and Reinhard Grisshammer MRC Laboratory of Molecular Biology Hills Road Cambridge UK 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 puri cation 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. Purifcation to homogeneity was achieved in three steps including ligand affinity chromatography based on the antagonist xanthine amine congener. The purifed hA2aR fusion protein bound 3H ZM241385 with a Kd of nM and an average Bmax of 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. Keywords adenosine A2a receptor 3H ZM241385 G-protein-coupled receptor maltose-binding protein fusion functional solubilization. Adenosine is a paracrine modulator of cell function that is important for local regulatory .

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