Translational Control of Membrane Proteins Rituximab antibody was obtained from Genentech Manufac- turing, anti-His from Roche, anti-FLAG M2 from Sigma and anti- Prokineticin 1 from Novus Biologicals. buffer B with detergent and 300 mM imidazole. These samples were then dialyzed against buffer B and detergent to remove imidazole. Cloning and Expression The cDNA for human CD20, RA1c, EG-VEGFR1, and patched 1 were sub-cloned, using standard molecular biology techniques, into a pBR322-derived plasmid containing the b- lactamase gene and tRNA genes for three rare E. coli codons (argU, glyT and pro2). A short Uni (MKHQHQQ) and 79 amino acid LE sequence were added to the N-terminus of the membrane proteins and an octa-His sequence was placed at the C-terminus to aid in detection and purification. A thrombin cleavage site (LVPRGS) has been placed after the LE leader to allow removal of the leader sequence. Gene transcription is under control of the phoA, phac or tphac promoter. Gene expression was induced by dilution of a saturated LB carbenicillin culture into C.R.A.P. phosphate limiting media [39]. The culture was then grown at 30uC for 24 hours or the specified time for the phoA promoter. The Pho regulon generally turns on approximately 7–8 hours post dilution when the cell density reaches an optical density at 600 nm (OD600) of 2. For the tphac promoter induction, cultures were induced at OD600 of 2 with 1 mM IPTG for 6 to 10 hours or the time specified. Human CD20 cysteine residues 111 and 220 were mutated to serines by site directed mutagenesis to improve protein behavior during purification. Protein Purification To determine protein location by detergent solubility, cells were lysed in buffer B (20 mM Tris, pH 7.5, 300 mM NaCl) by sonication and the membrane fraction was isolated by centrifu- gation. The membrane pellet was then re-suspended in buffer B and 1% Fos-Choline 12 (FC-12) and extracted overnight at 4uC. Samples were then centrifuged at 100,0006g for 1 hour and the supernatants collected. As necessary, the detergent soluble fraction was further purified using Ni-NTA Phynexus (San Jose, CA) pipette tips according to the manufacture’s instructions. For large-scale extraction, cells were re-suspended in 10 mL/g buffer A (20 mM Tris, pH 7.5, 5 mM EDTA) and centrifuged at 12,0006g for 30 min. The cell pellet was then re-suspended in buffer B (see above), lysed by cell disruption using a microfluidizer (Microfluidics Corp., Newton, MA) and centrifuged at 125,0006g for 1 hour. To extract the membrane protein from the cell membrane, the pellet was re-suspended in buffer B, FC-12 was added to 1% and the solution was stirred overnight at 4uC. The next day, the detergent insoluble fraction was pelleted by ultracentrifugation at 125,0006g for 1 hour. The supernatant was loaded onto a Ni-NTA Superflow (Qiagen Inc. Valencia, CA) column pre-equilibrated in buffer B containing 5 mM FC-12 (buffer C). The column was washed with 10 column volumes of 20 mM imidazole in buffer C and eluted with buffer C with 250 mM imidazole. All purification steps through column loading were performed at 4uC. Eluent fractions containing CD20 were concentrated and loaded onto a Superdex 200 column (Amersham Biosciences, Piscataway, NJ) pre-equilibrated in buffer C. The his-tagged human CD20 was further purified over a 5 mL HiTrap HP Q (Amersham Biosciences, Piscataway, NJ) column prior to gel filtration. For LE-CD20, the LE leader was removed by thrombin before size exclusion chromatography. For detergent exchange, samples were passed over a Superdex 200 column in 0.1% dodecyl maltoside, 150 mM NaCl, 20 mM HEPES, pH 7.2. Alternatively, samples were bound to a small Ni- NTA column, washed with buffer B and detergent and eluted in Density Gradient Centrifugation A discontinuous sucrose gradient was generated by layering 1.75, 1.4 and 0.8 M sucrose solutions of Buffer D (150 mM NaCl and 20 mM HEPES, pH 7.2) in centrifuge tubes. E. coli membrane proteoliposome preparations were prepared by cell disruption in buffer D (10 mL/g) containing 1 mM EDTA. The insoluble fraction was isolated by centrifugation at 38,0006g for 1 hour. The supernatant was discarded and the pellet was re- suspended in Buffer D containing 0.25 M sucrose. This re- suspension was mixed with 1.9 M sucrose solution, resulting in final concentration of 1.75 M sucrose. 1 mL of this mixture was then placed at the bottom of a centrifuge tube and equal volumes of the 1.4 M and 0.8 M sucrose solutions were layered above. Samples were centrifuged for 1 hour at 100,0006g. Samples in 200 mL aliquots were removed from the top of the tube and analyzed by SDS-PAGE, transferred to nitrocellulose and probed with horseradish peroxidase conjugated anti-his antibody. ELISA Assays 96 well plates were coated overnight at 4uC with 100 mL of CD20 at 1 mg/mL in PBS with solubilizing detergent diluted to below its critical micelle concentration. Plates were then washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked for 45 minutes at room temperature with 200 mL of PBST containing 0.5% BSA (blocking and assay buffer). Plates were washed again three times with PBST and probed with the primary antibody. 150 mL of rituximab at 60 mg/mL in assay buffer was added to the appropriate wells and three fold serial dilutions were performed in the subsequent wells by taking 50 mL from the first well and mixing with 100 mL of assay buffer in the next and subsequent wells to a final concentration of approximately 2 ng/ mL. After 90 minutes incubation at room temperature, the plates were washed with PBST and bound rituximab was detected with 100 mL of horseradish peroxidase conjugated goat anti-human F(ab9)2 (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) diluted 1:2,000 in assay buffer, washed six times with PBST and developed with 100 mL/well of TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD) mixed according to the manufacturer’s instructions. The reaction was halted by the addition of 100 mL/well of 1.0 M phosphoric acid and the absorbance was measured at 450 nm using a plate reader. Reduced and alkylated CD20 samples were prepared by reduction with 10 mM DTT and alkylation by addition of 25 mM iodoacetamide. The reaction was halted by a further addition of 100 mM DTT. Following each step, the reaction was allowed to proceed for 30–60 minutes at room temperature at pH 8.0. EC50 values were determined by 4-parameter fit of the data. FACS Analysis For preparation of spheroplasts, 5 OD600 mL of induced cells were recovered from expression media by centrifugation for 5 minutes at 5,000 rpm in a tabletop rotor (4,0006g). The supernatant was discarded and the pellet was re-suspended in 350 mL of ice-cold spheroplast preparation buffer A (50 mM Tris- HCl, pH 8.0, 25% sucrose, 100 mg/mL lysozyme, 67 mL/mL complete EDTA free protease inhibitor tablet in 2 mL deionized H2O) and the solution was diluted with 700 mL of ice-cold 1 mM EDTA. This mixture was allowed to incubate for 10 minutes at room temperature. 50 mL of 0.5 M MgCl2 was then added to PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35844