Figure 1.
Expression of a Gag/Δpp65 fusion protein in yeast.
(A) Schematic outline of Gag/Δpp65 before and after in vivo assembly into chimeric yeast VLPs. (B) SDS-PAGE and anti-pp65 immunoblot of crude extracts from yeast expressing either Δpp65 (lane 1), Gag (lane 2), or Gag/Δpp65 (lane 3). To ensure in vivo translation initiation of N-terminally truncated Δpp65 (24.9 kDa), a methionine residue was added to the N-terminus of Δpp65 [M, prestained PAGE Ruler, Fermentas].
Figure 2.
Sedimentation profile and electron microscopy of Gag/Δpp65 expressed in yeast demonstrate in vivo assembly into isometric VLP chimeras.
(A) Western analysis of chimeric Gag/Δpp65 particles and natural L-A virions assembled in yeast and purified by ultracentrifugation through a linear sucrose gradient. Aliquots of each gradient fraction were separated by SDS-PAGE and probed with monoclonal anti-pp65 and polyclonal anti-Gag, respectively [GP, gradient pellet; M, full range rainbow marker, Amersham]. (B) Electron micrograph of sucrose-gradient-purified Gag/Δpp65 after negative staining with uranyl acetate/methyl cellulose [magnification 150,000; arrows indicate Gag/Δpp65 particles].
Figure 3.
Gag/Δpp65 expressed in yeast assembles into VLP chimeras strongly activating CD8+ memory T cells in human whole blood.
(A) Western blot of Gag/Δpp65 (lane 1) and Gag (lane 2) expressed in yeast and partially purified as sucrose cushion pellet after ultracentrifugation. Aliquots (20 µl each) of the indicated VLP preparation were subjected to SDS-PAGE followed by Coomassie-Blue staining and western analysis probed with anti-Gag and/or anti-pp65 [M, full range rainbow marker, Amersham]. (B) Frequencies of antigen-specific CD4 and (C) CD8 T cell activation after stimulation by sucrose cushion-purified yeast Gag and Gag/Δpp65 particles. Activated T cells were identified as CD69/IFN-γ double-positive lymphocytes by flow cytometry. Antigen samples were added to whole blood from HCMV seropositive donor 1. A VLP-free sample containing PBSE buffer was included as negative control (NC), a lysate from HCMV-infected fibroblasts served as positive control (PC). The threshold of significant T cell responses (0.05% of counted lymphocytes [15]) is indicated as dashed line.
Figure 4.
Purified Gag/Δpp65 chimeras induce an extensive human CD8 T cell response.
(A) SDS-PAGE and anti-pp65 immunoblot of sucrose gradient purified Gag and Gag/Δpp65 particles expressed and assembled in yeast [Coomassie-Blue staining and BSA (2.7 µg) were used for semi-quantitative signal detection; M, full range rainbow marker, Amersham]. (B and C) Whole blood cells from three HCMV seropositive donors were stimulated by the addition of either Gag or Gag/Δpp65 (5 µg each), and specifically activated CD4 (B) and CD8 (C) T cells were quantified as CD69/IFN-γ double-positive lymphocytes by flow cytometry. A lysate from HCMV-infected fibroblasts served as positive control (PC), whereas a lysate from noninfected fibroblasts, a VLP-free buffer sample as well as blood cells from HCMV seronegative donor 5 were used as negative controls (NC). The threshold of significant T cell responses (0.05% of counted lymphocytes [15]) is indicated as dashed line.
Figure 5.
Chimeric Gag/K28α particles displaying the toxic α-subunit of the K28 virus toxin assemble into yeast VLPs that induce an in vivo antibody response in rabbit.
(A) SDS-PAGE and Coomassie-Blue staining of recombinant Gag/K28α particles expressed and assembled in yeast and purified by sucrose gradient centrifugation. (B) Western analysis of the α/β heterodimeric K28 virus toxin probed with a rabbit polyclonal antiserum raised against chimeric Gag/K28α VLPs assembled in yeast. Positions of the heterodimeric K28 toxin (α/β) and its tetrameric derivative [α/β]2 that forms spontaneously under conditions of a non-reducing SDS-PAGE are indicated (PI, pre-immune serum).
Figure 6.
GFP expression and purification via recombinant yeast VLPs.
(A) schematic outline of a Gag/GFP fusion (GTXG) for in vivo VLP assembly and purification of the model protein GFP. The particular function of each domain within the protein fusion is indicated. (B) SDS-PAGE and anti-GFP western analysis of recombinant GTXG particles assembled in yeast and purified by sucrose gradient centrifugation. (C) Release of GFP from GTXG particles by factor Xa cleavage [rGFP, recombinant GFP; M, full range rainbow marker, Amersham]. (D) Single-step purification of GFP obtained after factor Xa treatment and ammonium sulfate (AS) precipitation (S, supernatant; P, pellet) by hydrophobic interaction chromatography on a HIC column. Samples were separated by SDS-PAGE and probed with anti-GFP and/or anti-T7. (E) Coomassie-Blue staining of pooled GFP-containing HIC fractions after Amicon ultrafiltration through a 10 kDa cut-off membrane [rGFP, recombinant GFP; M, full range rainbow marker, Amersham].
Figure 7.
Chimeric yeast VLPs expressing bacterial esterase (EstA) function as recyclable bioreactor and show efficient substrate conversion.
(A) Electron micrograph of recombinant Gag/EstA particles prepared from yeast were purified by sucrose gradient centrifugation, negatively stained with uranyl acetate/methyl cellulose and subsequently used for electron microscopy (magnification 340,000). (B) Linear correlation between the 4-nitrophenol concentration of up to 1 mM and its absorption at 405 nm. (C) Kinetics of Gag/EstA-driven hydrolysis of 4-nitrophenylacetate (280 µM) to 4-nitrophenol and acetate at 25°C in PBS50 buffer (pH 7.0). (D) Coomassie-Blue staining and western analysis of Gag/EstA particles before and after cata-lysis and recycling by ultracentrifugation. BSA (1 and 3 µg) was used as loading control [M, full range rainbow marker, Amersham]. (E) Specific activity of chimeric Gag/EstA particles before and after recycling.