title: Milligram production and biological activity characterization of the human chemokine receptor CCR3.
abstract: Human chemokine receptor CCR3 (hCCR3) belongs to the G protein-coupled receptors (GPCRs) superfamily of membrane proteins and plays major roles in allergic diseases and angiogenesis. In order to study the structural and functional mechanism of hCCR3, it is essential to produce pure protein with biological functions on a milligram scale. Here we report the expression of hCCR3 gene in a tetracycline-inducible stable mammalian cell line. A cell clone with high hCCR3 expression was selected from 46 stably transfected cell clones and from this cell line pure hCCR3 on a milligram scale was obtained after two-step purification. Circular dichroism spectrum with a characteristic shape and magnitude for α-helix indicated proper folding of hCCR3 after purification. The biological activity of purified hCCR3 was verified by its high binding affinity with its endogenous ligands CCL11 and CCL24, with K D in the range of 10(-8) M to 10(-6) M.
authors.affiliation: State Key Laboratory of Heavy Oil Processing, China University of Petroleum Huadong, Qingdao, Shandong, PR China.
authors.forename: Mingqing
authors.initials: M
authors.lastname: Wang
authors.forename: Baosheng
authors.initials: B
authors.lastname: Ge
authors.forename: Renmin
authors.initials: R
authors.lastname: Li
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Lao
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
mesh_terms: D002087:Butyrates / Q000494:pharmacology; D060846:Cell Engineering; D054413:Chemokine CCL11 / Q000737:chemistry; D054423:Chemokine CCL24 / Q000737:chemistry; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D010957:Plasmids / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D017510:Protein Folding; D054397:Receptors, CCR3 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection
chemical_list: D002087:Butyrates; C515456:CCL11 protein, human; C515484:CCL24 protein, human; C515350:CCR3 protein, human; D054413:Chemokine CCL11; D054423:Chemokine CCL24; D008024:Ligands; D054397:Receptors, CCR3; D011993:Recombinant Fusion Proteins; D013752:Tetracycline