Identification of the purified protein with Western blots using monoclonal antibodies All the protein fractions in the two-step purification of Trx- hCCR3 were identified using Western blot detection using rabbit anti-human CCR3 monoclonal antibody (Figure 7B). The elution from the Ni2+ chelating column purification of Trx-hCCR5 was also identified by Western blot detection using rabbit anti-human CCR5 monoclonal antibody (Figure 7E). However, in addition to that of Trx-hCCR5 (Figure 7E), a smaller fragment showed positive reaction with the hCCR5 monoclonal antibody. We speculated that the small fragment is perhaps a cleavage from the N-terminus of hCCR5 with an apparent molecular weight ,25 kDa with the abnormal migration for membrane proteins in SDS-PAGE and the monoclonal antibody recognizing the cytoplasmic domain of hCCR5. A high-efficiency E. coli expression system for high-level production of GPCRs Biophysical and structural studies of membrane proteins require milligram quantities of proteins. To obtain such an amount is one of the bottlenecks for membrane protein structural determinations [7]. Therefore, significant effort and resources have been invested in obtaining quantity and quality of membrane proteins. Recently, several human GPCRs were expressed in E. coli, but in most cases, at a low level, except for the human NK1 receptor (hNK1R) which was expressed in a large amount, albeit as inclusion bodies [66]. Functional chemokine receptors CCR5 and CXCR4 have been isolated on a small scale using biosensor technology, suitable to be used only for analysis of ligand/receptor interactions [71]. Human adenosine A2a receptor has been functionally expressed in E. coli as fusion with periplasmic maltose-binding protein (MBP) at the N-terminus. A yield of 1.5 mg was obtained from 100 g of wet cells [22]. Rat neurotensin receptor is another GPCR expressed in E. coli as dual fusions with MBP at N-terminus and Trx at C-terminus. It yield 10 mg from 200 L of culture [23]. Our study is the first report that each of four medically important human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1 has been expressed as a membrane-associated protein and purified in milligram amount using synthetic genes expressed in E. coli expression systems, as N-terminal thioredoxin fusion proteins. Although no ligand binding assays were carried out to study the function and expression of the Trx-hCRs in membranes, it does not necessarily mean that they are not functional [72]. The Trx-hCRs were detected by their respective monoclonal antibodies, on immunoblots, were highly stable in detergent solution and our CD data indicated that the Trx-fusion CRs were correctly folded. Screens for crystallization have been initiated. The genes of hCRs encoded using human codon frequency should have no obvious effects on expression in E.coli, since it has been reported that even optimization of codon frequency for E. coli will not have a significant effect [73,74]. More likely, the selection of suitable promoters and plasmids is pre-requisite for regulated expression of heterologous genes. The pBAD-DEST49 vector containing the arabinose araBAD operon and the HP-thioredoxin fusion partner are central factors in this system and showed high capacity for expression of GPCRs. The pBAD vectors are particularly suitable for overexpression of membrane proteins, and possess general applicability which is proven by the fact that members of four families of membrane proteins were overex- pressed using such vectors and yielded at least 1 mg purified proteins per liter of culture [75]. This is also in part due to the use of the 1,200 fold repressible PBAD promotor, the transcription Study Human Chemokine Receptor from which can be regulated in a dose-depended manner by L- arabinose from the concentration of 0.00002% to 0.2% [52]. Overexpression of heterologous membrane proteins fused to a highly expressed soluble protein at the N-terminus has proven very successfully [19,23,24,74,76]. In the present system, HP-thior- edoxin functions as a fusion partner and starts the translation of the target proteins. It may also facilitate to solubilize the GPCR in our system. It remains to be seen if this system can be generalized for other membrane proteins when more and more membrane proteins are produced using this HP-thioredoxin system. There is evidence that thioredoxin (Trx) possesses cytokine-like and chemokine-like activities [77], which hints at the possibility of specific interactions between the chemokine receptors and their fusion partner Trx. Therefore, we speculate that its function is deeply related to the stability of these chemokine receptor by means of particular interaction helping to stabilize conformation of hCRs. Purification of non-fused hCCR3 expressed by pEXP- hCCR3 in C41 has been carried out, however, the proteins eluted in the Ni2+ chelating column were not from the membrane fractions, but from inclusion bodies (data not shown); although the truncated form of Trx-hCCR5 was identified using Western blot (Figure 7E), it could not be separated in the gel filtration step. Using a low-copy-number plasmid with a moderately strong promoter and lower temperature during production has proven successful for membrane proteins [20]. Grisshammer and Tate reported that low-level bacterial expression could lead to higher degree of integration of the protein to the cytoplasmic membrane of E. coli, which is taken as the rate-limiting step for overexpression of membrane proteins [7]. Thus this simple, rapid and easy to scale-up system offers a strategy for high-level production of GPCRs for biophysical and structural studies. Materials and Methods Materials Reagents for PCR, TOPO Cloning and Gateway Cloning, vectors, E. coli strains One Shot Top10 chemically competent cells (Top10) and BL21(DE3)-STAR-pLysS, DNA Ladder, SDS-PAGE gels and protein standards were purchased from Invitrogen (Carlsbad, CA) unless otherwise noted. OverExpress Competent Cells, including C41(DE3), C43(DE3), C41(DE3)-pLysS and C43(DE3)-pLysS, were obtained from Lucigen (Middleton, WI). All detergents were purchased from Anatrace (Maumee, OH). Nitrocel- lulose membranes were purchased from Bio-Rad (Hercules, CA). Complete protease inhibitor cocktail tablets, EDTA-free, were purchased from Roche (Mannheim, Germany). Rabbit anti-human CCR3 monoclonal antibody (Ab 32512), rabbit anti-human CCR5 monoclonal antibody (Ab32048) and goat anti-rabbit IgG (HRP) were purchased from Abcam (Cambridge, MA). Mouse His6-tag monoclonal antibody and goat anti-mouse IgG/M HRP were obtained from Novagen (Gibbstown, NJ). Protein purification materials were purchased from GE Healthcare Life sciences, Uppsala, Sweden. All common chemicals were obtained from either Sigma (St. Louis, MO) or VWR International unless otherwise indicated. Liquid growth media used for E. coli culture were Luria-Bertani (LB) medium, Terrific Broth (TB) medium and 2YT medium, which were made referring to [78]. Corresponding media plates were made by adding 1.5% agar. The concentrations of antibiotics used in the media and media plates were 100 mg/ml ampicillin, 50 mg/ml kanamycin, and 34 mg/ml chloramphenicol, respec- tively. Additionally, 0.3 mM isopropyl-1-thio-b-D galactopyrano- side (IPTG) or 0.05% L-arabinose was added to make the media plates in the presence of inducer. PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4509