Highly-expressed human gene set used in codon usage optimization.

<p>Highly-expressed human gene set used in codon usage optimization.</p

Optimization of the <i>vpu</i> gene for expression in <i>E</i>. <i>coli</i>.

<p>Codon usage is presented by plotting the relative adaptiveness, <i>w</i>, values (a moving geometric mean with a 10-residue window) as a function of the residue number. Codon usage analysis of the native HIV-1 Vpu gene against the reference set of <i>E</i>. <i>coli</i> and <i>H</i>. <i>sapiens</i> are shown as red and blue traces. The codon usage of the <i>vpu</i> gene optimized for expression in <i>E</i>. <i>coli</i> is shown in black. The codon adaptation index (CAI, relative adaptiveness averaged over the entire length of a sequence) for each gene is shown as a broken line.</p

Size-exclusion chromatography of concentrated metal-affinity chromatography-purified PelB-VPU.

<p>Eluate from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172529#pone.0172529.g006" target="_blank">Fig 6</a> was subjected to SEC-FPLC. Sec resin: Superdex 200 10/300 GL; Flow-rate: 0.5 mL/min, Running buffer: 20 mM HEPES, pH 7.5, 250 mM NaCl, 5% glycerol and 0.02% βDDM. The minor peak at 8 mL corresponded to the void volume, and the major peak at 11.48 mL contained PelB-Vpu. The column was calibrated with cytochrome c (A), carbonic anhydrase (B), albumin (C), alcohol dehydrogenase (D), β-amylase (E) and blue dextran (F) and a standard curve was obtained as shown in the inset. The size of the Pel-B-Vpu- βDDM complex was estimated to be 315 kDa.</p

Vpu is inserted into the bacterial inner membrane with its C-terminal domain within the cytoplasm.

<p>Panel A depicts the possible outcomes when <i>E</i>. <i>coli</i> cells (I) and protoplasts (II III and IV) that express Vpu are digested with chymotrypsin alone (I-III) and in the the presence of Triton X-100 (IV). If the C-terminal domain of Vpu is protected by the inner membrane (I and II), it is expected to be resistant to chymotrypsin digestion, similar to the case of the cytoplasmic resident protein GroEL. If the C-terminal domain of Vpu is exposed at the periplasmic space of the cells (III), it is expected to be digested in the same way as AcrA. All three proteins should undergo chymotrypsin cleavage when the inner membranes are permeabilized by the addition of Triton X-100 (IV). <i>E</i>. <i>coli</i> cells (lanes 1 and 3) and protoplasts (lanes 2 and 4–8) were incubated without chymotrypsin (lanes 1, 2, and 7) or with the indicated concentrations of chymotrypsin (μg/mL); in the presence of 1% Triton X-100 (lanes 7 and 8) or its absence (lanes 1–6). Following treatments, cells were solubilized in the presence of SDS sample buffer and protein samples were resolved by SDS-PAGE followed by immunoblotting with antibodies against the periplasmic domain of the inner-membrane protein AcrA (Panel B, red arrow), cytoplasmic protein GroEL (Panel C, blue arrow) or Vpu C-terminal domain (Panel D, black arrow).</p

Co-immunoprecipitation of PelB-Vpu with CD4.

<p>Purified CD4 TMD + cytoplasmic domain fused to the amino end of MBP (CD4-MBP) was incubated with PelB-Vpu in βDDM micelles. The complex was captured by anti- MBP antibody-conjugated beads and pelleted by centrifugation and analyzed by SDS-PAGE followed by Coomassie staining (<b>A</b>) and immunoblot analysis with Vpu antibodies (<b>B</b>). Panel <b>A</b> depicts all proteins pulled down by only agarose beads (lane 1) and agarose beads conjugated with anti-MBP (lanes 2–5). Anti-MBP, CD4-MBP, MBP and Vpu are denoted by blue, green, red and black arrows respectively. Beads were incubated with purified proteins: CD4-MBP (lane 1), MBP and Vpu (lane 2), CD4-MBP (lane 3), Vpu lane 4), CD4-MBP and Vpu (lane 5).</p

Circular dichroism spectrometry analysis shows that PelB-Vpu is stable (A) at various temperatures up to 40°C and (B) across a wide spectrum of salt concentrations.

<p>Circular dichroism spectrometry analysis shows that PelB-Vpu is stable (A) at various temperatures up to 40°C and (B) across a wide spectrum of salt concentrations.</p

Protein analysis of the talon metal affinity chromatography purification of PelB-Vpu.

<p>Protein fractions were subjected to SDS-PAGE followed by silver staining (<b>A</b>) and immunoblotting (<b>B</b>). Detergent (βDDM)-soluble fraction containing PelB-Vpu (lane 1) was loaded onto a column containing Talon resin that was pre-equilibrated with 20 mM HEPES, pH 7.5, supplemented with 500 mM NaCl, 5 mM imidazole, and 0.02% βDDM. Unbound proteins in the flow through were collected (lane 2) and the column was washed consecutively with four different buffer solutions (20 mM HEPES, pH 7.5): W1 (lane 3, buffer plus 500 mM NaCl), W2 (lane 4, buffer plus 500 mM NaCl and 0.02% βDDM), W3 (lane 5, buffer plus 250 mM NaCl and 0.02% βDDM), and W4 (lane 6, buffer plus 250 mM NaCl, 0.02% βDDM and 10 mM imidazole). Pure PelB-Vpu was eluted with W4 buffer supplemented with 300 mM imidazole (lane 7, arrows).</p

Organization of the expression cassette within the plasmid pTM 875.

<p>Expression was driven by a T7 promoter as shown in (<b>A</b>). The Vpu gene is flanked by the PelB signal peptide at the N-terminus that directs the protein to the periplasmic space of the bacteria and facilitates integration into the inner membranes, and a 6x- histidine tag at the C- terminus to aid in purification. (<b>B</b>) shows the sequence of the PelB signal peptide (in orange), Vpu (in green) and histidine tag (in blue). Chymotrypsin cleavage sites are denoted by down-pointing arrows (⬇) for strong recognition sites and by an asterisk (<b>*</b>) for secondary sites.</p

Dynamic light scattering measurements show that PelB-Vpu is monodispersed.

<p><i>The complex of detergent and PelB-Vpu was detected as a narrow peak at 10 nm</i>. <i>A molecular weight of</i> 332.2 <i>kDa was predicted for the protein-detergent complex</i>. Monodispersity of PelB-Vpu (10 mg/mL) is shown by the heat map diagram (A) and a plot of frequency of each particle size estimated from multiple measurements (B).</p

Vpu fractionates with the water insoluble fraction and can be subsequently solubilized by detergents.

<p>Whole cell lysates of <i>E</i>. <i>coli</i> BL21s expressing Vpu were fractionated into non- aqueous (lane 1) and aqueous (lane 2) fractions by centrifugation. The non-aqueous fraction (pellets) were resuspended in extraction buffer containing various detergents (1% final concentration): βDM (lane 3), βDDM (lane 4), CHAPS (lane 5), OG (lane 6), LDAO (lane 7) and DPC (lane 8). Following overnight incubation at 4°C, detergent-soluble proteins were separated from insoluble proteins by centrifugation and fractionated by SDS-PAGE followed by immunoblot analysis using a Vpu-specific antibody. Vpu is indicated by a black arrow.</p