Figure 2. Improved cell growth and general accumulation of integral membrane proteins using a dually regulated promot- er. (A) Restricted E. coli growth in LB with the phoA-RA1c construct is relieved by using the tphac promoter, which reduces basal level expression. A 24-hour growth curve shows the empty pBR322 vector control (blue triangles), phoA-RA1c expression construct (green diamonds), tphac-RA1c expression construct (red circles) and phoA- EGFL7 as a non-membrane protein control (brown squares). (B) A representative western blot of RA1c expression from the phoA promoter is shown following induction by phosphate depletion when the cells reach approximately 2 OD600 (time 0). Maximum expression is reached within two hours post induction. By 6 hours, aggregation has begun and by twelve hours almost all the protein has moved from the monomer band to high molecular weight aggregate. Basal expression is shown after overnight growth in LB medium (LBON). The western blot was probed with an HRP coupled anti-his antibody. (C) A comparison of basal expression in LB of the GPCR proteins, RA1c and EG-VEGFR1, from the phoA and tphac promoters by western blot analysis. The phoA constructs show significant accumulation levels of the membrane proteins while the tphac constructs have reduced the accumulation to background levels. The arrow points to the monomer protein band. doi:10.1371/journal.pone.0035844.g002 accumulation of the two GPCRs was particularly striking and higher than expected for a modest change in translation initiation rates. Expression levels of these membrane proteins with the Uni leader were also compared to constructs with no leader. The lack of a leader resulted in very low expression compared to the Uni with CD20, RA1c and EG-VEGFR1, while Patched 1 expression was modestly higher than that observed with the Uni leader (Figure S3). However, because the translation initiation rates of the constructs without leaders are unknown, it is difficult to interpret these results. Translational Control of Membrane Proteins Figure 3. Improved integral membrane protein expression with the LE leader. (A) Comparison of the expression levels with the Uni and the LE leaders for multi-spanning membrane proteins CD20, RA1c, EG-VEGFR1 and Patched 1. Arrows point to the monomer protein bands for the two GPCRs. (B) The Uni leader has a higher translation rate than the LE leader at the beginning of the induction, but the rates reverse by the end of the induction. Relative translation rates were measured by pulse labeling cells expressing CD20 with 35S cysteine for five minutes as well as by assessing accumulation levels in whole-cell extracts by immunoblot with HRP conjugated anti-His antibody. The non- membrane protein EGFL7 was used as a control. doi:10.1371/journal.pone.0035844.g003 Control experiments fusing the native Met Patched 1 translation initiation region (TIR- first seven residues) to an unrelated protein EGFL7 show that the native Patched 1 TIR is extremely weak, and no translated protein could be detected in experiments similar to those shown in figure 3B (data not shown). The first 160 base pairs at the start of the Patched 1 gene are highly G/C rich (79%), and significant mRNA secondary structure would be expected to inhibit translation initiation at the planned start for the Met construct, and possibly the Uni construct. The most likely explanation for the minor expression observed from the native Met Patched 1 TIR in figure S3 is internal translation from Met152, which possesses a good Shine-Dalgarno just upstream. Translation from this residue is consistent with the observed molecular weight. Comparison relative translation rates In order to compare the relative translation initiation rates for the two leaders, CD20 synthesis rates were determined early in the induction. Cells were induced for 30 minutes after which samples were removed for western blot analysis of CD20 accumulation with each leader. The culture was then labeled with 35S cysteine for 5 minutes, and his-tagged CD20 was isolated by Ni-NTA resin. After separation by SDS-PAGE and transfer to nitrocellu- lose, CD20 was visualized either by anti-his western blot or autoradiography. Surprisingly, the results shown in figure 3B reveal that the Uni leader has a higher translation rate than the LE leader early in the induction. A similar experiment with the two PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35844