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. Results and Discussion PCR-based gene synthesis of human chemokine receptors Genes of hCCR5, hCCR3, hCXCR4 and hCX3CR1 were de novo synthesized from a set of overlapping DNA oligonucleotides using a two-step assembly/amplification PCR method [47]. Using the online program DNAworks http://helixweb.nih.gov/dnaworks [48], the gene sequences of hCCR5, hCCR3, hCXCR4 and hCX3CR1 were optimized using human codon preference (Figure S1). The full-length genes were parsed into 42 oligos with the maximum length of 45 nucleotides. Assembly PCR (PCR1) and amplification PCR (PCR2) were successfully carried out (Figure 1). The PCR1 products (Fig 1A) including the full-length genes were later specifically amplified in PCR2 (Fig 1B). The number of PCR cycles in PCR1 is very important for successfully producing the full- length gene fragments. This number in some degree depends on the number of oligonucleotides in the starting set, as was described by Baedeker [49]. However, this still is an empirical approach to obtain the best results for each gene. Subsequently, we cloned the 4 synthetic hCR genes into pCT-Blunt II-TOPO vector by TOPO Cloning. In the same manner, our group also successfully synthesized several genes including human tetraspanin membrane protein CD81 and human olfactory receptor 17-4, mouse olfactory receptors mOR23, mS51 and I7 [46,50,51]. The choice of expression vector systems Synthetic genes of hCRs were expressed in E.coli system using GatewayH Technology since it is the most commonly used system for heterologous protein productions. It is also convenient for plasmid construction and optimization of expression levels [7]. For rapid testing of protein expression and efficient purification, a His6-tag was fused to the C-terminus of the hCRs in the attB-PCR step. Finally, each synthetic hCR gene was independently subcloned into pEXP3-DEST vector and pBAD-DEST49 vector, for screening different expression systems. The high-copy-number pEXP3-DEST plasmid vector, which has a strong T7 promoter, is designed for in vitro protein production although it is also useful for expression in E.coli strains expressing the T7 RNA polymerase. The elements of hCR constructs in the pEXP3-DEST (pEXP-hCR) include a N- terminal His6-tag and a Lumio tag followed by a TEV recognition site, and an attB1, flanked fragment inserted by the Gateway Cloning technique (Figure 2A). The arabinose PBAD promoters, which provide tight, dose- dependent regulation of heterologous gene expression and inducible with L-arabinose, are particularly suitable for expression of membrane proteins [52]. The low-copy-number pBAD- DEST49 plasmid vector was used as a Gateway Cloning destination vector for the synthetic hCR genes. They feature the fusion partner HP-thioredoxin (Trx-), followed by an Enterokinase recognition site (EK) and attB-flanked fragment containing the gene of interest (Figure 2B). The fusion partner HP-thioredoxin (His-Patch thioredoxin) is modified from E.coli thioredoxin by E32H and Q64H mutations to form a ‘‘patch’’ together with His8, which binds metal ions, to purify proteins on metal-chelating resins. Furthermore, thioredoxin functions as a translation leader for high-level expression and in some cases, to improve the solubility and stability of the fused membrane proteins [53]. Study Human Chemokine Receptor Figure 1. Analysis of PCR-based gene synthesis of human chemokine receptors. Full-length genes of hCCR5 (1,056 bp), hCCR3 (1,065 bp), hCXCR4 (1,056 bp) and hCX3CR1 (1,065 bp) were de novo synthesized using a two-step assembly/amplification PCR method and examined on 1% TAE agarose gel electrophoresis by staining with 2 mg/ ml of ethidium bromide (EB). Samples (5 ml) were mixed with 1 ml of 66 DNA loading buffer per lane. 1 Kb DNA ladder is also shown. A) Detection of assembly PCR (PCR1) products of each hCR. B) Detection of amplification PCR (PCR2) products of the full-length genes from PCR1. doi:10.1371/journal.pone.0004509.g001 Selections of E.coli host strains Unlike soluble proteins, membrane proteins, in general, have to be inserted into host membranes to assume their correct conformation. This often introduces toxicity to host cells during overexpression. Several E.coli strains have been developed to reduce such toxic effects associated with high-level expression of membrane proteins. However, the choice of the most suitable host strains for expression of a particular membrane protein is still empirical. Therefore, to attain high-level production, milligrams per liter of cell culture of functional membrane proteins, a systematic optimization of the expression system need to be carried out in a combinatorial manner including: 1) transcription promoters, 2) expression vectors, 3) host cell strains, 4) growth media conditions, 5) inducer concentration, 6) timing of induction, 7) duration of post induction, and 8) growth temperature of post induction [7]. Host strain screening for expression of pEXP-hCRs, was carried out with 5 E.coli strains: 1) BL21(DE3)-Star-pLysS (BL21), 2) C41(DE3) (C41), 3) C43(DE3) (C43), 4) C41(DE3)-pLysS (C41pLysS) and 5) C43(DE3)-pLysS (C43pLysS). The strain C41 and C43 were derived from BL21 to achieve high-level production of heterologous membrane proteins, reportedly for solving the problem of plasmid instability during the expression of toxic recombinant membrane proteins [54,55]. We compared LB-agar plates I to II, BL21 and C41 cells that cannot form colonies in the presence of inducer IPTG (II), typical for expression of membrane proteins (Figure 3A). However, it does not necessarily suggest that the foreign gene is toxic to host strains PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4509