For over 50% of these determined membrane protein structures, the proteins were purified from naturally abundant sources. In contrast, less than 10% of soluble proteins were from natural sources, and over 90% were produced as recombinant proteins [14]. Therefore, future efforts need to focus on procedures for high-level heterologous expression of membrane proteins, effective solubilization in the presence of surfactants and purification for crystallization screening [15,16]. Heterologous expression of functional GPCRs has been accomplished in Escherichia coli, yeast, insect and mammalian cells, but with varying degree of success because of differences in host cell environment [17,18]. Insect and mammalian cell expression systems have been most frequently employed for pharmacological development. For biophysical and structural studies that require large amounts of GPCRs, yeast and E. coli systems are attractive for their ease of large-scale production but have been used with varying success [7]. There is no universal system suitable for GPCR production. The approach to achieving high-level production must often rely on empirical solutions for each particular GPCR. E.coli is a widely used system for heterologous protein production and is often perceived as an easy way to produce large amounts of eukaryotic proteins because of its simplicity of use and the availability of various expression plasmids and E.coli strains which have been reported to support high-level protein production. Furthermore, the short time required for plasmid construction and expression allows rapid optimization of purification schemes and inexpensive material for purification [7,17,19]. However, reports of GPCR expression in E.coli have shown extremely low yields [20]. Many factors may affect the efficiency including 1) codon usage efficiency, 2) translational initiation, 3) mRNA stability, 4) stability of the expressed protein, and 5) toxicity of the expressed protein in the host cells [7]. Several methods have been developed to overcome these problems and increase the protein yield. Notably, fusing target proteins to a highly expressed bacterial protein has proven particularly effective for improving the expression level of membrane proteins in E. coli [21]. Several GPCRs have been functionally expressed in E.coli and purified in milligram quantities as fusion proteins [22,23,24]. Solubilization and stabilization of membrane proteins using detergents are the first critical steps in purification membrane proteins, and constitute a bottleneck for the structural biology of membrane proteins [25,26]. Membrane proteins require a membrane-like environment to maintain their correctly folded structures and functions during and after purification. Detergent micelles provide such environments surrounding the hydrophobic domains of membrane protein and keeping them soluble in an aqueous environment, and thus are widely used for solubilization and purification of membrane proteins [27]. Generally, the suitable detergent or detergent mixture should solubilize the target protein most effective, keep it stable, and prevent its self- aggregation. However, due to individual differences between membrane proteins, the choice of detergent or detergent mixture for a particular protein cannot be predicted. Therefore, a systematic approach is required to select the optimal detergents to achieve solubilization and stabilization of each target protein. Chemokines are a family of small chemotactic cytokines (,8– 14 kDa), which function as chemo-attractants for various types of leukocytes and play a vital role in host defense mechanisms and lymphocyte development [6,28,29,30,31]. Chemokines are divid- ed into four subfamilies based on the arrangement of two N- terminally conserved cysteine residues: a- or CXC chemokines (recently named CXC ligands, CXCL), b- or CC chemokines (CCL), c- or C chemokines (lymphotactin, XCL) and CX3C Study Human Chemokine Receptor chemokines (fractalkine, CX3CL) [6,29,30,31]. The biological functions of chemokines are mediated by binding to cell surface chemokine receptors, which belong to the superfamily of G- protein-coupled receptors (GPCRs) [29]. Chemokines and their receptors are implicated in a wide range of human diseases, including acute respiratory distress syndrome, allergic asthma, psoriasis, arthritis, multiple sclerosis, cancer metastases, athero- sclerosis, and AIDS infection. [6,28,32,33]. We selected 4 human chemokine receptors in this study. Human chemokine receptors CXCR4 (hCXCR4, termed Fusin) and CCR5 (hCCR5) have been identified as principal co- receptors, besides CD4, for entry of human immunodeficiency virus 1 isolate (HIV-1) into target cells. Specifically, CXCR4 is used for T cell line-tropic strains (X4), CCR5 for macrophage- tropic strains (R5), and another HIV-1 strains (R5X4), also named dual-tropic primary isolate, utilizes both CXCR4 and CCR5 as entry co-receptor [34,35]. Additionally, CXCR4 and its specific ligand SDF-1 were shown to be associated with several cancers, such as breast cancer, head and neck cancer, small-cell lung cancer and non-small-cell lung cancer [36,37]. Human chemokine receptor CCR3 (hCCR3) is highly expressed on eosinophils and binds multiple chemokine ligands such as eotaxin (CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26), and MCP-4 (CCL13) with high affinity [38]. CCR3 also function as co-receptor for some isolates of HIV-1 and HIV-2 [39]. Furthermore several clinical studies suggest that eotaxin/CCR3 plays a pivotal role in allergic diseases, including allergic asthma, rhinitis, and atopic dermatitis [31,40]. Human chemokine receptor CX3CR1 (hCX3CR1), the specific receptor for the fractalkine (FKN), is expressed on inflammatory leukocytes such as natural killer cells, monocytes and T lymphocytes, and mediates both cell-adhesive and migratory behavior of leukocytes, in addition, it is expressed at particularly high levels in neurons and microglia in the brain [41,42]. CX3CR1 was also 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]. Altogether, these 4 chemokine receptors represent attractive targets for intervention against such major diseases as AIDS, cancers and allergic diseases. Recently, certain chemokine receptor antagonists derived from chemokine peptides and small molecules were developed to block chemokine receptors [45]. However, the mechanisms of receptor interaction with their ligand and drugs remain poorly understood for lack of detailed molecular structures of chemokine receptors. Here we report high-level productions of human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1. We synthesized the genes using a two-step assembly/amplification PCR method, then inserted them into pEXP3-DEST and pBAD- DEST49 vectors for high-level expression screening in E.coli. We first systematically screened and selected optimal: 1) E.coli host strains, 2) growth media, 3) induction and 4) temperature for protein production. After optimizing the expression of these chemokine receptors, we proceeded to systematically screen 96 detergents for use in purification procedures. The Fos-choline series, particularly FC14, was found to be most effective. This finding corroborates the previous work by Cook et al., who screened a wide range of detergents and found FC14 to be the optimal detergent for solubilizing and stabilizing another human GPCR, olfactory receptor hOR17-4 [46]. We also carried out secondary structural analysis of the purified protein and confirmed their molecular identities using monoclonal antibodies and some PLoS ONE | www.plosone.org 2 February 2009 | Volume 4 | Issue 2 | e4509