Figure 6. Expression and folding of CD20 in the E. coli inner membrane. Cell surface expression and orientation of CD20 was assessed from spheroplasts of E. coli cells expressing either Uni-CD20 (blue), LE-CD20 (green) or an empty control vector (red) treated with Alexa-488 conjugated anti-CD20 antibody to the extracellular loop of CD20 and analyzed by flow cytometry. doi:10.1371/journal.pone.0035844.g006 High-expression yields Extraction of proteins using native detergents confirmed the high expression levels with the LE leader and the phac promoter. Western blots suggest that approximately 90% of CD20, RA1c or EG-VEGFR1 are extracted in Fos-Choline 12 (FC12); however, Patched 1 is largely resistant to extraction in this detergent. In addition, LE-CD20 can be extracted in a mixture of FC12 and dodecyl maltoside (DDM) detergents (Figure S5) further indicating a native like conformation of this protein in the membrane [34]. The detergent FC12 has demonstrated excellent properties for solubilizing the E. coli membrane [35], and Fos-Choline detergents and FC12 have shown favorable properties for the isolation of eukaryotic membrane proteins [36] including GPCRs [37]. Expression of LE-CD20 can be detected in coomassie stained whole cell extract, while the GPCR proteins require additional enrichment using Ni-NTA resin (Figure 7A). Single step IMAC purification of all three proteins provide 2 to 10 mg of protein per liter at greater than 90% purity as estimated from coomassie stained gels (Figure S6). Large and small-scale isolations of CD20, RA1c, and EG-VEGFR1 (Table S1) show yields are reproducible within two fold. Quantification of LE-CD20 expression levels in whole cell extracts against a standard curve of purified LE-CD20 show total cellular expression levels to be 41 mg/L (Figure S7 and Methods S1), indicating 25% protein recovery after primary purification. We estimate recovery of EG-VEGFR1 and RA1c to be similar. The high LE-CD20 expression levels translate to 36105 molecules per cell – consistent with FACS data. Characterization of LE-CD20 Earlier functional expression and purification of CD20 demonstrated isolation of 10–20 mg of His-tagged protein from a gram of E. coli cells. For comparison, LE tagged human CD20, under the transcriptional control of the tphac promoter, was expressed in E. coli and isolated from cell membranes by IMAC affinity chromatography followed by thrombin cleavage of the LE leader and size exclusion chromatography. Representative samples of purified his-tagged human CD20 are shown in the SDS polyacrylamide gel in figure 7B. CD20 isolated in this relatively simple manner is over 95% pure with a final yield better than Translational Control of Membrane Proteins Figure 7. Protein expression, isolation and characterization. (A) CD20 (left most panel) could be seen in whole cell extracts, while RA1c and EG-VEGFR1 (right panel) required purification on Ni-NTA resin. (B) Coomassie-stained SDS gel of purified CD20. Lane 1) LE-CD20 non- reduced; 2) Uni-CD20 non-reduced; 3) Molecular weight markers: 200, 116, 97, 66, 55, 36.5, 31, 21.5 14.4 6 kDa; 4) LE-CD20 reduced; 5) Uni- CD20 reduced. (C) Activity of Uni and LE human CD20. Activity of the isolated proteins was assayed using the conformation specific antibody rituximab. Binding to LE-CD20 (solid black line, solid squares), Uni-CD20 (dashed line, solid circles), reduced and alkylated LE-CD20 (negative control) (solid gray line, open squares), and (control) PBS (solid black line, open circles). The curves for rituximab binding were determined from a 4-parameter fit and the LE leader was cleaved from CD20 before analysis. doi:10.1371/journal.pone.0035844.g007 5 mg/L of protein in shake-flasks or 1 mg/g cells. The protein migrates with an apparent molecular weight of approximately 35 kDa under reducing conditions, which is in reasonable agreement with the calculated molecular weight of 33 kDa. In both reducing and non-reducing SDS-PAGE, purified LE-CD20 shows significantly fewer contaminating protein bands and less protein aggregate than Uni-CD20, consistent with the improved expression properties of LE-CD20. To confirm proper folding and processing of LE-CD20, the presence of the disulfide bond in the extracellular domain of CD20 was evaluated using the conformation specific antibody rituxmab [33] in the ELISA assay described previously [15]. In this assay, rituximab binds LE-CD20 with an EC50 of 0.77 nM (Figure 7C). This affinity is tighter than the binding of rituximab to control Uni-CD20 of 3.1 nM and in reasonable agreement with previously reported data [15]. As an additional control, LE- CD20 was reduced and alkylated and assayed for rituximab binding. This procedure eliminates rituximab binding, thus PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35844