Purification and characterization of recombinant human endothelin receptor type A

• Published in: Protein expression and purification Vol. 84; no. 1; pp. 14 - 18
• Main Authors: Lee, Kwangkyu, Jung, Yuna, Lee, Jae Youl, Lee, Won-Kyu, Lim, Dongbin, Yu, Yeon Gyu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2012
• Subjects: Cell Membrane - metabolism; Chromatography, Affinity; Endothelin; Endothelin-1 - metabolism; Escherichia coli; Escherichia coli - metabolism; Expression; G-protein; GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism; Histidine - chemistry; Histidine - metabolism; Humans; Interaction; Protein Binding; Protein Conformation; Purification; Receptor; Receptor, Endothelin A - chemistry; Receptor, Endothelin A - isolation & purification; Receptor, Endothelin A - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Viral Matrix Proteins - chemistry; Viral Matrix Proteins - metabolism; G-protein; Endothelin; Purification; Receptor; Expression; Interaction
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2012.04.011

► An overexpression system of human endothelin receptor (ETA) was established. ► Bacterially expressed recombinant ETA was purified to homogeneity. ► Recombinant ETA composed of mainly α-helices. ► Recombinant ETA interacts specifically with ET-1 and Gαq. Human endothelin receptor type A (ETA) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ETA, we have expressed recombinant ETA as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ETA 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-ETA appeared as an oligomer and presented mainly as an α-helical structure. The protein also showed specific binding to endothelin-1 (ET-1) and the alpha subunit of Gq protein with apparent KD values of 17 and 20nM, respectively. An antagonist of ETA, bosentan, prevented the interaction between p9-ETA and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ETA has a competent conformation for interactions with ET-1 and the alpha subunit of Gq protein.