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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Butyrates / pharmacology
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Cell Engineering
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Chemokine CCL11 / chemistry
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Chemokine CCL24 / chemistry
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Gene Expression
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HEK293 Cells
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Humans
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Kinetics
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Ligands
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Plasmids / chemistry*
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Promoter Regions, Genetic
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Protein Binding
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Protein Biosynthesis / drug effects
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Protein Folding
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Receptors, CCR3 / biosynthesis*
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Receptors, CCR3 / chemistry
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Receptors, CCR3 / genetics
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Receptors, CCR3 / isolation & purification
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Tetracycline / pharmacology
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Transfection
Substances
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Butyrates
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CCL11 protein, human
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CCL24 protein, human
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CCR3 protein, human
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Chemokine CCL11
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Chemokine CCL24
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Ligands
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Receptors, CCR3
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Recombinant Fusion Proteins
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Tetracycline
Grants and funding
This work was financially supported in part by the National Key Basic Research Program of China (2012CB518000), the National Natural Science Foundation of China (No. 31000377, 21073236, 21033005), Shandong Province Natural Science Foundation (No. ZR2010BQ016), the program for New Century Excellent Talents in University of Ministry of Education of China (NCET-10-0815), the Natural Science Foundation for Distinguished Young Scholar of Shandong Province (No. JQ201008), and the Fundamental Research Funds for the Central Universities (No. 11CX04030A). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.