Purification and characterization of recombinant human endothelin receptor type A

► 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...

Full description

Saved in:
Bibliographic Details
Published in:Protein expression and purification 2012-07, Vol.84 (1), p.14-18
Main Authors: Lee, Kwangkyu, Jung, Yuna, Lee, Jae Youl, Lee, Won-Kyu, Lim, Dongbin, Yu, Yeon Gyu
Format: Article
Language:English
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
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:► 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.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2012.04.011