title: Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5.
abstract: Chemokine receptors play pivotal roles for immune cell recruitment to inflammation sites, in response to chemokine gradients (chemotaxis). The mechanisms of chemokine signaling, especially the initiation of the intracellular signaling cascade, are not well understood. We previously identified a cytoplasmic protein FROUNT, which binds to the C-terminal regions of CCR2 and CCR5 to mediate chemokine signaling. Although large amounts of purified protein are required for detailed biochemical studies and drug screening, no method to produce recombinant FROUNT has been reported. In this study, we developed a method for the production of recombinant human FROUNT. Human FROUNT was successfully expressed in Escherichia coli, as a soluble protein fused to the folding chaperone Trigger Factor, with a cold shock expression system. The purified FROUNT protein displayed CCR2 binding ability without any additional components, as demonstrated by SPR measurements. A gel filtration analysis suggested that FROUNT exists in a homo-oligomeric state. This high-yield method is cost-effective for human FROUNT production. It should be a powerful tool for further biochemical and structural studies to elucidate GPCR regulation and chemokine signaling, and also will contribute to drug development.
authors.affiliation: Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Esaki
authors.forename: Yuya
authors.initials: Y
authors.lastname: Terashima
authors.forename: Etsuko
authors.initials: E
authors.lastname: Toda
authors.forename: Sosuke
authors.initials: S
authors.lastname: Yoshinaga
authors.forename: Norie
authors.initials: N
authors.lastname: Araki
authors.forename: Kouji
authors.initials: K
authors.lastname: Matsushima
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Terasawa
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D054390:Receptors, CCR2 / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein
chemical_list: C515334:CCR2 protein, human; D029968:Escherichia coli Proteins; C503214:NUP85 protein, human; D028861:Nuclear Pore Complex Proteins; D054390:Receptors, CCR2; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase