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
Authors
김기선김은경신상철김남혁유연규
Keywords
EP4; GPCR; Overexpression; PGE2; G protein; Purification
Issue Date
2021-03
Publisher
Biochemistry and Biophysics Reports
Citation
VOL 25, 100871
Abstract
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.
URI
http://pubs.kist.re.kr/handle/201004/73032
ISSN
2405-5808
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KIST Publication > Article
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