title: High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1.
abstract: Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis.
authors.affiliation: Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Ren
authors.forename: Daoyong
authors.initials: D
authors.lastname: Yu
authors.forename: Baosheng
authors.initials: B
authors.lastname: Ge
authors.forename: Brian
authors.initials: B
authors.lastname: Cook
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
authors.forename: Shuguang
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000073883:CX3C Chemokine Receptor 1; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D054397:Receptors, CCR3 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019707:Receptors, Chemokine / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled
chemical_list: C515350:CCR3 protein, human; D000073883:CX3C Chemokine Receptor 1; C095235:CX3CR1 protein, human; C527276:CXCR4 protein, human; D054397:Receptors, CCR3; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D019707:Receptors, Chemokine; D043562:Receptors, G-Protein-Coupled