confirming the proper formation of the CD20 extracellular disulfide bond. Ligand binding to LE-EG-VEGFR1 To demonstrate proper folding and function of one of the GPCRs, we evaluated ligand binding to LE-EG-VEGFR1 (RA1c has no known ligand). EG-VEGF (Prokineticin 1) was incubated with E. coli membrane proteoliposomes prepared from negative control cells and cells expressing LE-EG-VEGFR1 fused to a FLAG epitope at either the N or the C terminus. These membranes were extensively washed, pelleted by centrifugation and analyzed by SDS-PAGE and developed by immuno-blot using an antibody to EG-VEGF. As shown in figure 8, the EG-VEGF ligand binds to LE-EG-VEGFR1 membrane proteoliposomes, indicating at least some population of the receptor is properly folded. Although experimental conditions limit exact quantitation of the amount of ligand bound to the receptor, we estimate the amount of receptor bound EG-VEGF in these experiments at 26103 molecules/cell, from a series of known concentrations of the ligand. Based on our results for receptor expression and recovery (Table S1 and Figure S7), we estimate the receptor at approximately 96103–46104 molecules/cell. Accounting for the loss of correct orientation of the receptor in the membrane following generation of proteoliposomes, we estimate that 10–40% of the receptors are able to bind ligand. Discussion The expression of eukaryotic multi-spanning membrane proteins in E. coli is particularly difficult compared to the relative ease of producing cytoplasmic and secreted proteins. A number of efforts have been undertaken in different labs to identify and overcome the expression barrier with this class of proteins. This work includes the use of special bacterial strains [12], reduced transcription [13], proteomic analysis upon induction [8] and a variety of different affinity tags [14]. However, with the exception of a recent report involving the directed evolution of a GPCR that resulted in greater expression and stability [9], accumulation of these proteins per cell remained about the same. Additionally, the underlying molecular limitation of expression has remained elusive. Our study focused on the relationship between translation levels and the expression or accumulation of these membrane proteins in Figure 8. Ligand binding to the GPCR, LE-EG-VEGFR1. E. coli membrane proteoliposomes were treated with thrombin to remove the LE-leader and incubated overnight at 4uC with EG-VEGF in PBS. Pelleted membranes were separated by SDS-PAGE and developed by immuno- blot using an anti-EG VEGF antibody. Samples are: lane 1) pBR322 negative control; 2) LE-EG-VEGFR1, N-terminal FLAG; 3) LE-EG-VEGFR1, C-terminal FLAG. The location of EG-VEGF (molecular weight 9 kDa) is indicated by an arrow. doi:10.1371/journal.pone.0035844.g008 Translational Control of Membrane Proteins E. coli. Since translation levels are largely determined by the translation initiation rate, we began by comparing the expression of four mammalian multi-spanning membrane proteins fused to two previously described leaders, the Uni and the LE. The resulting expression per cell of membrane proteins with the two leaders varied by 1–2 orders of magnitude – a much larger than expected difference considering that both leaders were thought to have similar high translation rates. Subsequent analysis revealed that the LE leader, which produced much higher levels of membrane protein expression, actually had a translation initiation rate that was several fold lower than that of the Uni leader. A detailed look at the expression profile shows that the Uni leader with its stronger translation rate does indeed outpace the weaker translating LE leader very early in the induction. However, after approximately 30 minutes, membrane protein expression with the Uni leader slowed significantly with no further increase in protein accumulation. By contrast, the LE leader membrane protein expression and accumulation continued without change for several hours. This allowed the more slowly translating LE leader membrane proteins far greater total production than the initially highly translating Uni leader membrane protein. All four highly expressed mammalian proteins with the LE leader are membrane associated upon cell fractionation, and inserted with a native like structure in the cell membrane. We analyzed the orientation of one of the GPCRs, EG-VEGFR1. The N-terminus of this receptor is orientated towards the periplasm while the C-terminus is cytoplasmic as is expected for proper insertion. The receptor also shows binding to its ligand, EG- VEGF. Additionally, CD20 has the correct orientation in the membrane based on FACS analysis and rituximab antibody binding. The effect of translation levels on multi-spanning membrane protein expression can be quite significant and this needs to be understood at the molecular level. The most likely explanation for our observations is a potential bottleneck at the level of membrane targeting, presumably by the SRP. Translation at too high of a rate would be expected to overload the more limited co- translational secretory pathway in E. coli and quickly lead to a halt in translation as we observe with the Uni leader. A more moderate level of translation seen with the LE leader avoids an overload of this pathway, allowing for membrane targeting and insertion over longer periods of time. The halt in translation observed with the Uni leader soon after promoter induction suggests that there is a mechanistic membrane protein targeting- translation coupling that is maintained in the cell, although, the exact molecular nature of this coupling remains to be determined. Optimizing integral membrane protein accumulation could potentially be controlled at the level of transcription to achieve the desired translation rate. The promoter sequence could be modified to provide a specific transcriptional strength, or alternatively repressor controlled promoters could be induced with suboptimal inducer concentrations. This later approach, however, requires the deletion of inducer transporters [38] and limits host strain selection. Materials and Methods All E. coli experiments used the 58F3 strain which was derived from W3110 and has the following genotype Dfhu (DtonA) Dlon galE rpoHt (htpRts) DclpP lacIq DompTD(nmpc-fepE) DslyD. All detergents were obtained from Anatrace, Inc., Maumee, OH. Unless otherwise mentioned, all chemicals were obtained from Sigma-Aldrich, St. Louis, MO. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35844