stabilize the cell membrane and the mixture was incubated on ice for 10 minutes. To block non-specific binding, the spheroplasts were pelleted at 5,000 rpm for 5 minutes in a tabletop centrifuge, gently re- suspended in 0.5 mL of ice-cold 10% fetal bovine serum in PBS and incubated on ice for 10 minutes. Spheroplasts were stained by addition of Alexa 488 conjugated anti-CD20 antibody at a concentration of 10 mg/mL followed by incubation at room temperature for 1 hour with mild agitation. Spheroplasts were pelleted as before and washed three times with 500 mL of PBS. Cells were analyzed on an EPIC-XL fluorescently activated cell sorter with the gating area adjusted for the size of the E. coli cells. 35S Pulse-Labeling Cultures were induced for 30 minutes (14 hours for the late time point) with 1 mM ITPG at an OD600 of 2 and pulsed with 35S cysteine for 5 minutes. SDS was added to a final concentration of 2% to stop the labeling and then heated immediately at 95uC for 15 minutes to lyse the samples. The samples were then diluted with 2% FC-12 in PBS to bring down the SDS concentration to 0.2% so that they could be loaded onto a Ni-NTA spin column (Qiagen) and purified using a standard protocol provided by Qiagen. Eluates were separated by SDS-PAGE, transferred to nitrocellulose and exposed to a film. Supporting Information Table S1 Primary Protein Recovery. Summary of protein yields after IMAC affinity purification from small-scale, 100 mL and large-scale, greater then 1 L expression. (TIF) Figure S1 Restricted E. coli growth and small colony size formation following cell transformation with a multi-spanning membrane protein construct. Basal pro- tein expression from the phoA promoter is deleterious to cell growth. (a) vector control (b) phoA-RA1c expression construct uninduced (c) tphac-RA1c expression construct uninduced. (TIF) Figure S2 Nucleotide sequence of the phoA, phac and tphac promoters. (a) The phoA promoter showing the pho box and 210 sequences underlined (b) The dually regulated phac promoter showing the introduced lac operator underlined (c) The tphac promoter showing the addition of the l to transcriptional terminator upstream of the phac promoter. PhoA 210 sequence and Pho Box (blue), Lac operator (red), Lambda transcription terminator (brown), ATG translation start (green). (TIF) Figure S3 Membrane protein expression without a leader. Comparison of the expression levels with the Uni and leaderless (Met) constructs for multi-spanning membrane proteins CD20, RA1c, EG-VEGFR1 and Patched 1. Arrows point to the monomer protein bands for the two GPCRs. (TIF) References 1. Egea PF, Stroud RM, Walter P (2005) Targeting proteins to membranes: structure of the signal recognition particle. Curr Opin Struct Biol 15: 213–220. 2. Luirink J, von Heijne G, Houben E, de Gier JW (2005) Biogenesis of inner membrane proteins in Escherichia coli. Annu Rev Microbiol 59: 329–355. 3. Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, et al. (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433: 377–381. Translational Control of Membrane Proteins Figure S4 N and C-terminal FLAG epitopes of LE-EG- VEGFR1 are accessible to anti-FLAG antibody. Membrane proteoliposomes were prepared from E. coli expressing either N or C terminal FLAG tagged LE-EG-VEGFR1. Samples are: lane 1) pBR322 negative control; 2) LE-EG-VEGFR1, N-terminal FLAG; 3) LE-EG-VEGFR1, C-terminal FLAG; 4) pBR322 negative control; 5) LE-EG-VEGFR1, N-terminal FLAG; 6) LE-EG- VEGFR1, C-terminal FLAG. Samples for lanes one, two and three were treated with 1% Triton X-100 prior to incubation with anti-FLAG antibody. Samples for lanes four, five and six were treated with antibody in the absence of detergent. (TIF) Figure S5 Extraction of LE-CD20 from the cell mem- brane. Samples of E. coli membrane with expressed LE-CD20 were treated with a ratio of detergents from 1% FC-12 to 1% DDM. Lane 1) 1% FC-12; 2) 0.75:0.25; 3) 0.5:0.5; 4) 0.25:0.75; 5) 1.0% DDM. Membrane samples were extracted with detergent over night and CD20 was detected using an anti-His HRP conjugated antibody. (TIF) Figure S6 Representative gels of membrane proteins following large-scale purification over immobilized nickel column. Samples were detected by coomassie staining following separation on 4 to 20% SDS-PAGE. Samples are: lane 1) LE-CD20; 2) Molecular weight marker; 3) LE-EG-VEGF-R1; 4) LE-RA1c; 5) Molecular weight markers. Each sample lane contains 15 mg of protein. Molecular weights of the protein standards are shown on side of the figure. (TIF) Figure S7 LE-CD20 is expressed at high levels in E. coli. Total cellular level of LE-CD20 was determined by comparison to a standard curve generated with the purified protein. Based on the average OD600 of 3.0 for the LE-CD20 culture, total expression is 41 milligrams per liter of culture. Representative data from two independent measurements is shown. *Lane quantitation was determined using Licor-700. (TIF) Methods S1 Methods for quantitation of LE-CD20 expression levels in E. Coli. (DOC) Acknowledgments We thank Christoph Spiess for his critical reading of the manuscript and his helpful suggestions during its preparation. Author Contributions Conceived and designed the experiments: HSK JAE CB AH JB GF CVL RLV DGY. Performed the experiments: HSK JAE CB AH JB GF CVL RLV DGY. Analyzed the data: HSK JAE CB AH JB GF CVL RLV DGY. Contributed reagents/materials/analysis tools: HSK JAE CB AH JB GF CVL RLV DGY. Wrote the paper: HSK JAE DGY. 4. Powers T, Walter P (1997) Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor. Embo J 16: 4880–4886. 5. Walter P, Blobel G (1983) Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and comple- mentary DNA probe. J Cell Biol 97: 1693–1699. 6. Mason N, Ciufo LF, Brown JD (2000) Elongation arrest is a physiologically important function of signal recognition particle. Embo J 19: 4164–4174. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35844