membrane space, general transmembrane protein toxicity [8] and stability of the protein sequence itself [9]. Since the area of plasma membrane per volume in a eukaryotic cell is smaller than the area of plasma membrane per volume in a prokaryotic cell, simply based on cell size, it is unlikely that the amount of membrane is a limiting factor in protein expression. Likewise, since several proteins, the KcsA potassium channel [10], and bacteriorhodopsin [11], among others, can be expressed at several milligrams per gram of cell mass, it is unlikely that protein crowding in the plasma membrane is a limiting factor in expression. Previous attempts to improve membrane protein expression in E. coli have relied on selective screening to identify random mutations in specific bacterial strains [12,13]. With few exceptions, improvements were limited to bacterial proteins and rarely resulted in increased expression per cell. Attempts to address expression problems with simple N or C terminal tags have had limited success [14] while evaluation of various promoter systems has also shown similar modest improvement. Our study focused on determining the influence translation levels have on the expression of eukaryotic multi-spanning membrane proteins in E. coli. Using different leaders to control translation initiation, we show that translation initiation rates determine both initial induction rates and total protein accumu- lation. High translation rates quickly lead to a halt in synthesis while more moderate rates allow for high levels of accumulation over an extended period of time. Results Expression of membrane proteins Earlier work assessed the expression of CD20, a marker of human B-cells with four alpha-helical membrane domains [15]. That study demonstrated isolation of milligram quantities of CD20 in a native like conformation from the bacterial membrane, following expression from a vector previously described for E. coli protein production. In the current study, we attempted to extend this work to three new candidate proteins: the human G protein coupled receptors (GPCRs), RA1c [16,17] and EG-VEGFR1 [18,19,20] with 7-TM domains, and the 12-TM transport like protein Patched 1 [21,22]. Topology diagrams and molecular weights of the candidate proteins in their native state are shown in figure 1. These proteins were chosen solely based on their biological roles or potential as therapeutic targets. The three candidate genes were inserted into the original expression vector under the transcriptional control of the phoA Figure 1. Topology diagram of model human proteins as expressed in a mammalian cellular plasma membrane. The predicted molecular weights for these proteins without post-transla- tional modification are: CD20, 33.0 kDa; EG-VEGFR1, 44.8 kDa; RA1c, 35.5 kDa and Patched 1, 160.5 kDa. doi:10.1371/journal.pone.0035844.g001 Translational Control of Membrane Proteins promoter. In addition, each gene contains nucleotide sequences encoding a small seven amino acid MKHQHQQ (Uni) leader to provide an efficient translation initiation. Induction of CD20 and Patched 1 by means of phosphate limitation resulted in a stable level of protein expression over time, detectable by anti-his western (data not shown). However, both GPCR constructs had a significant toxic effect on the host as demonstrated by the size of the bacterial colonies (Figure S1). In addition, expression of either GPCR was problematic and variable due to the poor growth in even transcription-repressed conditions (Figure 2A). Further, we observed a striking pattern in the expression time course for both proteins. Both monomer and dimer forms of the two GPCRs were stable for about four hours post induction, after which time the proteins were transformed into high molecular weight aggregates. This can be clearly seen for RA1c in figure 2B. The time course of this transition suggests the proteins are correctly membrane inserted initially, but become highly aggregated over time. Basal level transcription controlling Toxicity from basal expression of the GPCR constructs in E. coli created significant experimental variability, which complicated any controlled study. To reduce basal transcription, we inserted the lac operator [23,24] at the +1 position of the existing phoA promoter [25,26]. The resulting phac promoter requires both phosphate starvation as well as the addition of the lac inducer, isopropyl b-D-1-thiogalactopyranoside (IPTG) for full induction (Figure S2). Partial induction levels can be achieved by manipulating each regulatory element individually in a lac repressor iQ strain. To further suppress possible upstream cryptic promoters, the l to transcriptional terminator [27] was introduced upstream of the phac to create the tphac promoter. Subsequent work has shown that the phac and the tphac promoters behave similarly. The phoA promoter in the GPCR expression constructs was replaced with the tphac promoter to assess whether or not basal level toxicity was still a problem. A comparison of the colony sizes of the E. coli host after transformation with the EG-VEGFR1 and RA1c plasmids suggested that the tphac promoter had significantly reduced the toxicity of both genes (shown for RA1c in Figure S1). The subsequent culture of these colonies in non-inducing conditions showed that there was no growth retardation with the tphac promoter as compared to those constructs with the phoA promoter (Figure 2A). Finally, basal accumulation of the GPCRs with the phoA promoter was reduced to background with the tphac promoter (Figure 2C). Alteration of translation levels To gain insights into the effects translation levels have on membrane protein expression, we initially attempted to increase translation levels to see if these membrane proteins could be forced into refractile bodies. As the Uni leader is optimized for translation initiation within the constraints of its coding sequence, we incorporated a new leader, which had previously been shown to result in exceptionally high translation levels, the trp LE. The trp LE was originally isolated as a fusion of the first 9 amino acids of the trp leader to distal parts of the E protein encoded in the trp operon [28]. We designed a leader based on the first 79 amino acids of the LE and fused this to the N-termini of each of the four studied proteins. Production of the four membrane proteins with the LE leader was compared to the original constructs using the smaller Uni leader. The tphac promoter was induced as noted earlier and a comparison of the leaders was made at 12 hours post IPTG addition. All four proteins with the LE leader showed a significant increase in production as shown in figure 3A. The increase in the PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35844