Purification and characterization of recombinant human endothelin receptor type A

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• 作者：Kwangkyu Lee^a ; Yuna Jung^b ; Jae Youl Lee^c ; Won-Kyu Lee^a ; Dongbin Lim^b ; Yeon Gyu Yu^a ; ^{ygyu@kookmin.ac.kr}
• 关键词：Endothelin ; Receptor ; G-protein ; Expression ; Purification ; Interaction
• 刊名：Protein Expression and Purification
• 出版年：2012
• 出版时间：July, 2012
• 年：2012
• 卷：84
• 期：1
• 页码：14-18
• 全文大小：397 K

文摘

Human endothelin receptor type A (ET_A) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ET_A, we have expressed recombinant ET_A as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ET_A fusion protein was highly expressed in the membrane fraction of Escherichia coli and purified to homogeneity by single affinity chromatography after solubilization with detergents. Purified p9-ET_A appeared as an oligomer and presented mainly as an ¦Á-helical structure. The protein also showed specific binding to endothelin-1 (ET-1) and the alpha subunit of G_q protein with apparent K_D values of 17 and 20 nM, respectively. An antagonist of ET_A, bosentan, prevented the interaction between p9-ET_A and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ET_A has a competent conformation for interactions with ET-1 and the alpha subunit of G_q protein.