Expression of the HFB library in <i>N</i>. <i>benthamiana</i>.

<p>(A) Amino acid sequences of the HFBI and the 8 novel hydrophobin fusion tags studied here. Conserved cysteine residues and disulphide bridges are highlighted. (B) Expression levels of fusion proteins determined by fluorometry. Letters indicate groups with significant difference (<i>p</i><0.05, <i>n</i> = 8 individual plants). Error bars indicate standard deviation. (C) Confocal microscopy images illustrate subcellular localization of the fusion proteins. A control sample infiltrated with only p19 showed no signal (image not show). The scale bar represents 5μm.</p

Comparison of HFBII and HFBIV to HFBI.

<p>(A) Accumulation in <i>N</i>. <i>benthamiana</i> (<i>n</i> = 8 individual plants). (B) ATPS with purified proteins to compare recovery rates (<i>n</i> = 3). Letters indicate significant difference (<i>p</i><0.05). Error bars indicate standard deviation.</p

Purification of HFB-fusion proteins by ATPS.

<p>(A) A schematic illustration of the process. (B) Purification from plant extract. The partition coefficient is determined by dividing concentration in surfactant phase by concentration in residue. Letters indicate significant difference (<i>n</i> = 4, <i>p</i><0.05) and error bars indicate standard deviation.</p

Accumulation of chloroplast-targeted, transiently-expressed rFaeG_ntd/dsc.

<p>Transient expression of the rFaeG_ntd/dsc protein via agroinfiltration in <i>Nicotiana benthamiana</i> leaves was examined by SDS-PAGE and staining (a), and immunoblot analysis (b). Lanes 1 and 2−5.0 µg of protein extract of leaves co-infiltrated with <i>Agrobacteria</i> carrying chloroplast-targeted rFaeG_ntd/dsc and the p19 viral suppressor of post-transcriptional gene silencing (1), or p19 alone as negative control (2). rFaeG_ntd/dsc is indicated with a black rhomb, higher bands likely correspond to rFaeG_ntd/dsc with partially cleaved transit peptide; Lane 3−0.5 µg purified F4_ad fimbriae as positive control, the F4 native FaeG is indicated with a black triangle; the ∼2 kDa difference in size of rFaeG_ntd/dsc (29 kDa) and the native FaeG (27 kDa) is due to the additional complementing fused domain.</p

Accumulation levels of rFaeG_ntd/dsc in transplastomic leaf tissue.

<p>(a) Samples of equal volume (4 µl) were prepared from crude extract fractions. Lane 1 - WT extract (negative control); lanes 2, 3 and 4 represent crude extract of 0.4 mg of leaf tissue, re-extracted pellet, and clarified extract, respectively, where clarified extract contains 5 µg TSP. The rFaeG_ntd/dsc yield was estimated using a standard curve (<i>R²</i> = 0.993) of known amounts of purified rFaeG_ntd/dsc (lanes 5 through 8∶2 µg, 1 µg, 0.5 µg and 0.25 µg, respectively). (b) No variation in rFaeG_ntd/dsc accumulation was observed in transplastomic clones (C1, C2) after dark (D) or after light (L) periods. Image is representative of sampling on three different days, 1 µg TSP was used per lane. WT =  untransformed control.</p

Purification of rFaeG_ntd/dsc from crude plant extract and quantification.

<p>(a) rFaeG_ntd/dsc was extracted from 5 g of mature transplastomic leaf tissue and purified. The initial volume of the extract was 50 ml; 3 µl of the extract from each step of the procedure were resolved by SDS-PAGE and stained. Lane 1 - Initial extract from leaf tissue, pH = 7.5; lane 2 - extract acidified to pH = 2 and centrifuged; lane 3 - clarified extract neutralized to pH = 7.4; Lane 4 - flowthrough from IMAC column; Lane 5 - wash with 20 mM imidazole; Lane 6 - elution of purified rFaeG_ntd/dsc; Lane 7 - 0.5 µg of BSA as loading control; kDa - protein molecular weight marker. (b) Purified rFaeG_ntd/dsc was quantified using densitometry. Dilutions of the purified rFaeG_ntd/dsc protein (lanes 1 through 7) were resolved in SDS-PAGE gel along with known amounts of BSA (lanes 8–14; 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 µg BSA, respectively) and stained. BSA bands were used for generation of a standard curve (<i>R²</i> = 0.987; <i>p</i> = 0.01) and extrapolating rFaeG_ntd/dsc concentration. kDa - molecular weight marker.</p

Chloroplast-produced rFaeG_ntd/dsc protein is recognized in F4 fimbriae-specific ELISA, partially polymerizes and specifically binds to the brush border of F4R+ small intestinal villi.

<p>(a) Both rFaeG_ntd/dsc and F4 fimbriae are recognized by a monoclonal anti-F4_ad fimbriae antibody in ELISA. (b) Purified F4 fimbriae (lane 1) and purified rFaeG_ntd/dsc (lane 2) were resolved under non-reducing conditions to assess polymerization. The F4 fimbriae sample displayed the formation of native FaeG polymers, number of subunits is indicated by stacked black triangles next to each band. Most of the rFaeG_ntd/dsc is present as monomers (denoted by black rhomb); formation of rFaeG_ntd/dsc dimers and trimers was also observed (two and three stacked black rhombs). (c) Adhesion of the rFaeG_ntd/dsc protein to the brush border of F4R+ small intestinal villi. Binding to the F4-specific receptors present on the apical surface of the epithelial cells, which line the brush border of F4R+ small intestinal villi is shown as a bright line on the edge of the sample, the result of excited FITC fluorochrome (indicated with white arrows, lower panel). rFaeG_ntd/dsc fails to bind to brush border of F4R− small intestinal villi. Images are representative of rFaeG_ntd/dsc adhesion to isolated villi of three F4R+ and two F4R− piglets. Bar: 50 µm.</p

Homoplastomic lines show normal phenotype.

<p>(a) A schematic representation of the chloroplast transformation cassette (pCT-rFaeG_ntd/dsc). The cassette was designed to integrate between the <i>trnI (tRNA-Ile)</i> and <i>trnA (tRNA-Ala)</i> genes of the tobacco plastome. The wild type (WT) plastome <i>trnI - trnA</i> region is shown at the bottom. Expected sizes of <i>Rsr</i> II-digested fragments are indicated. Thick black lines represent hybridization sites for the probe used in Southern blot analyses. IEE  =  intercistronic expression element with the Shine-Dalgarno sequence from the 5′ UTR of bacteriophage T7 gene 10 fused to the 3′ end; aadA  =  gene encoding aminoglycoside 3′ adenylyltransferase for spectinomycin resistance; T<i>psbC</i>  = 3′ UTR of <i>psbC</i> from white poplar plastome; P<i>psbA</i>  = 5′ UTR and promoter of tobacco <i>psbA</i> gene. <i>rfaeG_ntd/dsc</i>  =  gene encoding the rFaeG_ntd/dsc protein variant. T<i>rbcL</i>  = 3′ UTR of <i>rbcL</i> from white poplar plastome. (b) Phenotypes of mature transplastomic tobacco cv. I 64 plants transformed with pCT-rFaeG_ntd/dsc (1 and 2) were indistinguishable from WT plants (3). A one-meter ruler was photographed to the left of each plant as size reference. (c) Confirmation of homoplastomy. Southern blot analysis of total plant DNA from 2 independent transformants and 1 untransformed plant displayed a single band of the expected size.</p

Stability of rFaeG_ntd/dsc under simulated gastrointestinal conditions.

<p>Time course analysis of the stability of chloroplast-expressed rFaeG_ntd/dsc in simulated gastric fluid (SGF; a) and simulated intestinal fluid (SIF; b). rFaeG_ntd/dsc was present in similar amounts either as purified protein (“Purified”) or as lyophilized and powdered transplastomic leaf tissue (“Biomass”) and was visualized by western blotting. SGF digestion of leaf biomass was done at two different pH values: pH = 3.5 and pH = 4.5. SGF and SIF fluids with no substrate [SGF (−) and SIF (−), respectively] represent negative controls. The rFaeG_ntd/dsc band is indicated with an arrow.</p

Chloroplast-produced rFaeG_ntd/dsc inhibits the adhesion of F4+ ETEC to porcine small intestinal villi.

<p>Adhesion of F4+ ETEC to F4R− villi (a) and F4R+ villi (b), white arrows indicate bacterial cells. Bar: 50 µm. (c) Competitive inhibition of adhesion of F4+ ETEC to porcine small intestinal villi by the rFaeG_ntd/dsc protein or F4 fimbriae, determined at different protein concentrations. The data represent the mean ±SE (n = 4).</p