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Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins

Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E(2) (PGE(2)) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein...

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Autores principales: Kim, Nam Hyuk, Kim, Key-Sun, Shin, Sang Chul, Kim, Eunice Eunkyeong, Yu, Yeon Gyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749421/
https://www.ncbi.nlm.nih.gov/pubmed/33367116
http://dx.doi.org/10.1016/j.bbrep.2020.100871
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author Kim, Nam Hyuk
Kim, Key-Sun
Shin, Sang Chul
Kim, Eunice Eunkyeong
Yu, Yeon Gyu
author_facet Kim, Nam Hyuk
Kim, Key-Sun
Shin, Sang Chul
Kim, Eunice Eunkyeong
Yu, Yeon Gyu
author_sort Kim, Nam Hyuk
collection PubMed
description Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E(2) (PGE(2)) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of G(s) or G(i) proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4.
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spelling pubmed-77494212020-12-22 Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins Kim, Nam Hyuk Kim, Key-Sun Shin, Sang Chul Kim, Eunice Eunkyeong Yu, Yeon Gyu Biochem Biophys Rep Research Article Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E(2) (PGE(2)) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of G(s) or G(i) proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4. Elsevier 2020-12-16 /pmc/articles/PMC7749421/ /pubmed/33367116 http://dx.doi.org/10.1016/j.bbrep.2020.100871 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Kim, Nam Hyuk
Kim, Key-Sun
Shin, Sang Chul
Kim, Eunice Eunkyeong
Yu, Yeon Gyu
Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins
title Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins
title_full Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins
title_fullStr Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins
title_full_unstemmed Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins
title_short Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with G(α) proteins
title_sort functional expression of human prostaglandin e2 receptor 4 (ep4) in e. coli and characterization of the binding property of ep4 with g(α) proteins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749421/
https://www.ncbi.nlm.nih.gov/pubmed/33367116
http://dx.doi.org/10.1016/j.bbrep.2020.100871
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