Purification and characterization of recombinant human endothelin receptor type A

Protein Expression and Purification
Kwangkyu LeeYeon Gyu Yu

Abstract

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 α-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.

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Citations

Jan 17, 2013·Protein Expression and Purification·Jung-Hyun NaYeon Gyu Yu
Oct 7, 2014·Protein Science : a Publication of the Protein Society·Seong-Gu HanYeon Gyu Yu
Feb 6, 2016·Experimental & Molecular Medicine·Migyeong Jo, Sang Taek Jung
Nov 7, 2020·International Journal of Molecular Sciences·Man-Seok Ju, Sang Taek Jung
Oct 1, 2021·Experimental & Molecular Medicine·Man-Seok JuSang Taek Jung

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