Buffers for lysis and solubilization: Lysis buffer, 50 mM sodium phosphate pH 7.8, 200 mM NaCl, 100 mM KCl, 20% glycerol, 10 mM EDTA, 2 mM DTT, 1 mM PMSF, 50 mg/ml lysozyme, 20 mg/ml DNase I; Membrane Wash buffer, 50 mM sodium phosphate pH 7.8, 500 mM NaCl, 100 mM KCl, 20% glycerol, 10 mM EDTA, 1 mM PMSF; Solubilization buffer, 50 mM sodium phosphate pH 7.8, 200 mM NaCl, 100 mM KCl, 20% glycerol, 1% FC14, 1 tablet of protease inhibitor per 10 ml. Buffers for purification: Buffer A (Ni2+ chelating column binding buffer), 50 mM sodium phosphate pH 7.8, 200 mM NaCl, 100 mM KCl, 0.02% FC14, 25 mM imidazole; Buffer B (Ni2+ chelating column elution buffer), 50 mM sodium phosphate pH 7.8, 200 mM NaCl, 100 mM KCl, 0.02% FC14, 500 mM imidazole; and Buffer C, (gel filtration and purified protein storage buffer) 16PBS buffer (diluted from 106PBS buffer, EMD Chemicals, Gibbstown, NJ), 0.02% FC14. Buffers for Western blot and Dot blot: Blocking buffer, 16PBS, 10% skim milk, 0.1% Tween-20; Wash buffer, 16PBS, 0.5% skim milk, 0.1% Triton-X- 100. Other buffers: DNA loading buffer (66) was from Novagen; SDS Sample buffer (46), 250 mM Tris-HCL pH 6.8, 40% glycerol, 8% SDS, 0.004% Bromophenol Blue, 20% Mercapto- ethanol, dissolved in milliQ water and frozen at 220uC. Methods PCR-based gene synthesis. Human chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1 were selected in the GPCR structural biology program. Protein sequences of the hCRs were obtained from UniProt (Universal Protein Resource), namely human CCR5 (UniProt ID: P51681), human CCR3 (UniProt ID: P51677), human CXCR4 (UniProt ID: P61073) and human CX3CR1 (UniProt ID: P49238), as shown in Figure S1. For PCR- based gene synthesis, the receptor sequence was encoded using the human codon preference using the Dnaworks program (http:// helixweb.nih.gov/dnaworks) and parsed into an oligonucleotide set with the following parameters: 45 nt oligonucleotide length, 58uC annealing temperature, 19% of codon frequency threshold, 25 nM oligonucleotide, 10 mM Na+/K+ and 2.0 mM Mg2+. As shown in Figure S1, the encoded genes of hCRs consist of 1056 bp (hCCR5), 1065 bp (hCCR3), 1056 bp (hCXCR4) and 1065 bp (hCX3CR1), respectively. Each gene of hCRs was synthesized as a set of 42 oligonucleotides, synthesized at 50 mmolar scale in 96- well plate format by Integrated DNA Technologies (IDT, Coralville, IA). PCR-based gene synthesis was performed using a two-step assembly/amplification PCR protocol and designated PCR1 and PCR2. In PCR1, all 42 oligos were mixed and diluted 1:10 (5 mM for each oligos) in milliQ water as primers and also templates. PCR1 was executed following standard protocol [78], except 58uC anneal for 30 seconds, 72uC extension for 60 seconds, and 35 cycles, which are necessary for achieving full length of gene in PCR2. PCR2 was performed using diluted (1:100) PCR1 product as template and the two DNA oligos at the ends of the gene as primers, 72uC extension for 90 seconds and 25 cycles. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide (EB). PCR2 products were purified using QIAquick Gel Extraction Kit (QIAGEN) before used in TOPO Cloning, which is a blunt end subcloning used here for inserting the full-length synthetic genes into the pCR-Blunt II- TOPO vector for amplification and DNA sequencing. TOPO Cloning was performed following the manufacturer’s protocol. The plasmids with pTOPO-hCR inserts were purified using QIAprep Miniprep kit (QIAGEN) and sequenced. Gene recombination using Gateway Cloning Technology. There are three steps in Gateway Cloning: the Study Human Chemokine Receptor attB PCR product preparation, the BP Cloning reaction and the LR Cloning reaction (details see Instruction Manual of Gateway Cloning Technology, Invitrogen). The attB PCR product of synthetic genes of hCRs were produced by regular PCR using attB1-hCR-F (59- GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTA +24 nt of 59 terminal forward sequence of hCR) and attB2-Histag-hCR-R (59- GGGG ACC ACT TTG TAC AAG AAA GCT GGG TC TCA ATG GTG GTG ATG ATG GTG +24 nt of 39 terminal reverse sequence of hCR except for stop code) as primers, with corresponding pTOPO-hCR plasmids as templates. In the attB2-Histag-hCR-R primer, His6-tag codons were added at the C-terminus of hCRs for expression detection and protein purification. The PCR products were purified and used in the following BP Cloning reaction, which used pDONR- 221 vector to make Entry Clones of synthetic genes of hCRs. In the final step (LR Cloning reaction), each gene of hCR was cloned into pEXP3-DEST and pBAD-DEST49 for non-fused and thioredoxin-fused protein expression. All the reactions were performed following the product protocol, and all the Entry clones and Expression clones have been sequenced. Selection of suitable host strains and optimization of cell growth conditions. For selection of suitable host stains, pEXP- hCRs were transformed into E. coli strains BL21, C41, C43, C41- pLysS and C43-pLysS, respectively; while for pBAD-hCRs, the E. coli strains Top10 was recommended by manufacturer’s instruction. For toxicity detection and media type selection of pEXP-hCRs in the five E. coli strains, an equal amount of the transformation reactions were spread on LB-agar plates, LB-agar-IPTG plates, TB- agar-IPTG plates and 2YT-agar-IPTG plates, respectively, followed by overnight culture at 37uC. For pBAD-hCRs in Top10, toxicity detection and media type selection were performed in the same way, except that L-arabinose replaced the IPTG as inducer for gene expression. In addition, selection of media types from LB, TB and 2YT was also investigated in liquid media. The protocol was: colonies from LB-agar plates were selected and cultured in 5 ml of LB liquid medium overnight at 37uC with shaking; the next morning, 50 ml of overnight culture was inoculated in 5 ml of fresh LB, TB or 2YT liquid medium, respectively, and to culture was continued at 37uC with shaking while monitoring growth of the cultures by measuring the optical density at 600 nm (OD600); at OD600 of 0.6,0.8, the temperature was decreased to 16uC and after 20 minutes, the inducer was added (0.3 mM IPTG for pEXP-hCRs, or 0.05% L-arabinose for pBAD- hCRs). The concentration was monitored every 3 or 4 hours until harvest at 24 hours post induction. All plates and liquid media used here and in later experiments contained 100 mg/ml of ampicillin, except the strains with pLysS were 34 mg/ml of chloramphenicol was added to avoid loss of the pLysS plasmid. Optimization of gene expression. Five colonies from each of the control LB-agar plates (plates I or - in Fig 3A) were selected and separately cultured in 5 ml of LB medium overnight at 37uC with shaking. The following morning, each cell culture was separately inoculated (1:100) into 3 (for pEXP-hCRs) or 5 (for pBAD-hCRs) tubes containing 5 ml fresh TB medium each. Culture was continued at 37uC with shaking, until the concentration of cell cultures (OD600) arrived at 0.6,0.8. At this point, the temperature of the incubator was lowered to 16uC, and different concentrations of inducer were added to the culture. For pEXP-hCRs in the five strains, three concentrations at 0.1 mM, 0.2 mM and 0.3 mM of IPTG were tested for gene expression, while five different concentrations at 0.01%, 0.02%, 0.05%, 0.1% and 0.2% of L-arabinose were tested separately for the expression of each pBAD-hCRs in Top10. One ml of cell culture was withdrawn from each tube every 4 hours, pelleted at at 5000 g PLoS ONE | www.plosone.org 12 February 2009 | Volume 4 | Issue 2 | e4509