Kim, N.H. Kim, K.-S. Shin, S.C. Kim, E.E. Yu, Y.G. 2024-01-19T15:30:32Z 2024-01-19T15:30:32Z 2021-09-02 2021-03 2405-5808 https://pubs.kist.re.kr/handle/201004/117370 Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E2 (PGE2) 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 Gs or Gi 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. ？ 2020 The Authors English Elsevier B.V. Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with Gα proteins Article 10.1016/j.bbrep.2020.100871 1 Biochemistry and Biophysics Reports, v.25 Biochemistry and Biophysics Reports 25 N scopus 000646693800003 2-s2.0-85098158320 Article complementary DNA cyclic AMP DNA fragment G protein coupled receptor G protein coupled receptor alpha immunoglobulin G antibody polymer prostaglandin E receptor 4 unclassified drug alpha helix animal cell Article binding affinity binding kinetics binding site carboxy terminal sequence circular dichroism conformational transition controlled study Escherichia coli gene amplification heat tolerance mouse nonhuman protein binding protein conformation protein content protein degradation protein expression protein protein interaction protein purification protein secondary structure protein stability thermostability EP4 G protein GPCR Overexpression PGE2 Purification