Study Human Chemokine Receptor Figure 2. Schematic illustrations of 4 hCRs expressed as fusion proteins. In each synthetic hCR gene with a His6-tag was inserted at the C- terminus for expression, detection and protein purification, and each gene was cloned into pEXP3-DEST and pBAD-DEST49 vectors, respectively. A) Schematic representation of hCR cloned into pEXP3-DEST vector (simply pEXP-hCR). The translation start codon (ATG) followed by His6-tag, LumioTM –tag and TEV (TEV protease recognition site) are present in the vector pEXP3-DEST and fused at the N-terminus of hCR following the insertion of the C-terminally His6-tagged hCR at the attB1 site by the Gateway site-specific recombination reaction. B) Schematic representation of hCR cloned into pBAD-DEST49 vector (pBAD-hCR). Analogous to the pEXP-hCR vector, the ATG, HP-thioredoxin (Trx) and EK (Enterokinase recognition site) are present in the pBAD-DEST49 vector and fused at the attB1 site to the N-terminus of hCR, following Gateway protocols. doi:10.1371/journal.pone.0004509.g002 since the conditions for growth and induction may also influence the expression level. C43, C41pLysS and C43pLysS were able to form smaller colonies in the presence of IPTG (II) than those of in the absence of IPTG (I), which, although a promising sign, does not always guarantee successful expression of target genes [56]. Good growth was observed for pBAD-hCRs in Top10, which is the strain recommended in the manufacturers protocol since its deficient in araBADC. In the presence of L-arabinose (plates denoted with a +), Top10 cells formed colonies with smaller size than those on plates lacking L-arabinose (2) (Figure 3B). The expression levels of pEXP-hCRs in each of the 5 strains and of pBAD-hCRs in Top10 were studied from 5 selected colonies, each picked from the plates without inducer. 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. For the pEXP-hCCR5 construct, detectable expression was not achieved in any of these strains. Expression was in all cases induced using 0.25 mM IPTG in TB medium at 16–18uC for 12– 20 hours with shaking. The colonies of pEXP-hCRs in C43, C41pLysS and C43pLysS were larger than those in C41 and BL21 (Fig 3A). However, the gene expression levels in the former three strains appears to be lower than in the latter two strains. The, choice of the C41 over the BL21 strain for expression of pEXP-hCRs, was motivated by its better resistance to the toxic effects of heterologous membrane proteins in the host cells, as was described [54]. The expression of pEXP-hCRs in C41 was later studied by Western blot using mouse His6-tag monoclonal antibody. Selection of growth media The choice of growth media has a significant influence on cell growth and protein production. Thus, selection of suitable growth media and conditions for pEXP-hCR expression in each of the 5 E.coli strains was carried out by comparison of colonies between plates II, III and IV (Figure 3A). TB (Terrific Broth) medium plates (III) showed better growth conditions than other two types of media. For testing growth conditions for pBAD-hCRs in Top10 (Figure 3C), cell growth was further studied in liquid media. The culture density of pBAD-hCCR3 and pBAD-hCX3CR1 in Top10 significantly increased in TB medium as function of time, but no obvious change was observed for hCCR5 and hCXCR4. The cell density of pBAD-hCCR3 and hCX3CR1 in Top10 was able to reach OD600 7–8 units while those of hCCR5 and hCXCR4 only reached OD600 0.6–0.8, a 10-fold difference. The expression of hCCR5 and hCXCR4 thus appeared to induce a stronger toxic response in the host cells than hCCR3 and hCX3CR1. It is not surprising that different proteins have individual characteristics and behave differently in identical cells, which is a common observation in heterologous membrane protein expres- sion [7]. It is possible that media rich in nutrients in some cases may contribute to the damage from toxic effects of heterologous membrane protein expression. TB medium contains higher concentration of yeast extract and tryptone with additional glycerol (0.4% v/v), and the strong phosphate buffer, which make it suitable for long-term growth of cells. Thus, it was selected as the best medium for heterologous expression of hCRs. For pEXP- hCRs, each of the strains showed similar behavior i.e., after induction, the cell cultures reached high densities of 11–13 OD600 units, especially in TB medium (data not shown). In most cases, such high cell densities are indicative of a lower protein expression, protein expressed in inclusion bodies, or lower plasmid stability. Optimization of inducible hCR expression In order to optimize high-level hCR production, many variables must be systematically studied including timing, length of induction and culture temperature. To screen the high-yield production strains easily, a rapid and simple method is crucial. We used dot-blot detection with mouse anti-His6-tag monoclonal antibody since the expressed proteins all carried the His6-tag. As described in the previous section, pEXP-hCCR3 reached a higher level of expression than did pEXP-hCXCR4 or hCX3CR1, while no expression was detectable for pEXP-hCCR5 PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4509