Figure 4. Expression of hCRs detected with Western blot using mouse anti-His6-tag monoclonal antibody. SDS-PAGE was per- formed using NuPAGE 4–12% Bis-Tris gel (Invitrogen, 16 MES buffer). Lane M, His-tagged protein standard size markers (Invitrogen); Lane 1 through 4, proteins expressed by pBAD-hCCR5, hCCR3, hCXCR4 and hCX3CR1 in Top10, respectively; Lanes 5 through 8, proteins expressed by pEXP-hCCR5, hCCR3, hCXCR4 and hCX3CR1 in C41, respectively. doi:10.1371/journal.pone.0004509.g004 (Figure 4). The plasmid stability correlated with their expression level, e.g. higher stability for pEXP-hCCR3 (80%) than for pEXP- hCXCR4 (66%), hCX3CR1 (62%) and hCCR5 (50%). Expression of pBAD-hCRs in Top10 was similarly studied as for pEXP-hCRs. All the genes were expressed after induction with 0.05% L-arabinose in TB medium at 16–18uC from 2–24 hours. The expression of synthetic hCR genes expressed in pBAD-hCRs with the Trx fusion partner at the N-terminus (Trx-hCRs), are shown in Figure 4. The total expression levels of pBAD-hCRs are higher than those of pEXP-hCRs. Furthermore, the bands (lanes 1–4) show more proteins than those expressed by pEXP-hCRs (lanes 5–8), suggesting more stable and less aggregated expression of pBAD-hCRs in Top10 as compared to the expression of pEXP- hCRs in C41. The plasmids of pBAD-hCRs also show higher stability than pEXP-hCRs, i.e. 75% for pBAD-hCCR5, 93% for pBAD-hCCR3, 60% for pBAD-hCXCR4 and 80% for pBAD- hCX3CR1. Therefore pBAD-hCRs in Top10 were selected for expression of synthetic hCR genes. The apparent molecular weights of expressed hCRs and Trx- hCRs in SDS-PAGE gel are generally smaller than their Study Human Chemokine Receptor calculated molecular weight as shown in Table 1, which is not uncommon for membrane proteins, especially for GPCRs. The membrane proteins samples were mixed with SDS sample loading buffer and incubated at 37uC for 10 minutes before loading. Unlike for samples of soluble proteins, which need be boiled before loading, membrane protein samples cannot be boiled since boiling results in further aggregation. Therefore, membrane protein samples cannot be completely denatured only by SDS, thus have a more compact shape, which tends to faster migration in the SDS-PAGE gel, typically at ,70–85% of their expected molecular weight [57]. Systematic detergent screening for solubilization of Trx- hCRs Solubilization from the host cell membrane is absolutely crucial for successfully purification and stabilization of the heterologous membrane proteins [25,58]. The choice of a suitable detergent for solubilizing an individual membrane protein must be empirically determined. Even for membrane proteins in GPCR superfamily, different detergents have been used for solubilization of different GPCRs [59]. Therefore, a systematic approach for screening a wide range of detergents is essential for obtaining the expressed hCRs. Detergents generally are divided into 4 group depending on their chemical properties, namely, anionic (A), cationic (C), non- ionic (N) and zwitter-ionic (Z) [60]. To investigate the efficacy of various detergents for extraction of Trx-hCRs, 96 detergents were selected from the commercial Solution Master Detergent Kit and from literature. These detergents (Fig. 5) include members of maltosides and glucosides families, such as b-OG, DM and DDM, which have been successfully used to produce crystals; some generally used reagents for solubilization and purification of membrane proteins in many laboratories, such as Digitonin, TDAO, Triton and CHAPS; and some recently developed detergents, i.e. Cyclo-Fos series, Cymal series and Fos-Choline series. In addition, several detergent mixtures were also included in Figure 5 since they had proven effective in solubilization of some GPCRs [59]. The detergent concentrations used during extraction procedures were based on their critical micelle concentration (CMC) values. A concentration above the CMC is required to form micelles. For the majority of detergents, 1% was thus chosen as a standard concentration during the extraction trials, except for detergents with higher CMC, where ,2% was used. Detergents with much higher CMC (.2%) were not used since their detergent-protein ratios are hard to reach [25], except MEGA-8 (#52), which was chosen for completing the variety of detergents. Our laboratory has also been studying other GPCRs including human and mouse olfactory receptors since 2004. After extensive screening of a wide ranger of various detergents, Brian Cook found Table 1. Parameters of native and reconstructed human chemokine receptors. Amino Acids MW(kD)* pI** Amino Acids MW(kD)* pI** Amino Acids MW(kD)* pI** Amino Acids MW(kD)* pI** hCCR5 Native 352 pEXP3-hCRs 396 pBAD-hCRs 491 *MW, molecular weight. **pI, isoelectric point. doi:10.1371/journal.pone.0004509.t001 40.52 45.32 55.50 9.21 9.10 8.70 PLoS ONE | www.plosone.org hCCR3 355 399 494 41.04 45.84 56.02 6 8.49 8.49 7.07 hCXCR4 352 396 491 hCX3CR1 39.75 44.54 54.72 8.46 8.47 7.03 355 399 494 40.39 45.19 55.37 6.74 7.29 6.30 February 2009 | Volume 4 | Issue 2 | e4509