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.
Highlights
G-protein-coupled receptors (GPCRs) primarily function as cellsurface receptors responsible for the transduction of extra-cellular stimuli into intra-cellular signals by binding extra-cellular ligands including photons, ions, lipids, peptides, nucleosides, nucleotides, neurotransmitters and peptide hormones
Genes of hCCR5, Human chemokine receptor CCR3 (hCCR3), hCXCR4 and Human chemokine receptor CX3CR1 (hCX3CR1) were de novo synthesized from a set of overlapping DNA oligonucleotides using a two-step assembly/amplification PCR method [47]
The results suggested that pEXP3-hCCR3, hCXCR4 and hCX3CR1 were best expressed in C41 strain, to some extent, and expressed at a lower level, in C43 and BL21, but not expressed well in C41pLysS and C43pLysS
Summary
G-protein-coupled receptors (GPCRs) primarily function as cellsurface receptors responsible for the transduction of extra-cellular stimuli into intra-cellular signals by binding extra-cellular ligands including photons, ions, lipids, peptides, nucleosides, nucleotides, neurotransmitters and peptide hormones. CX3CR1 was identified as a co-receptor together with CD4 for entry of HIV-1 [43], and a role for CX3CR1-FKN-mediated inflammation has been suggested in various inflammatory diseases including vascular injury, atopic dermatitis and allergic airway diseases [44] These 4 chemokine receptors represent attractive targets for intervention against such major diseases as AIDS, cancers and allergic diseases. We carried out secondary structural analysis of the purified protein and confirmed their molecular identities using monoclonal antibodies and some cases, mass spectroscopy analysis To our knowledge, this is the first high-level production of human GPCR chemokine receptors in E.coli in milligram quantities sufficient for initiating structural studies
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