Figure 4
Co-purification and characterisation of co-expressed DENV E proteins. (a) Ni2+-NTA affinity chromatographic co-purification of E proteins co-expressed by the tetravalent P. pastoris clone. The solid blue curve represents the UV absorbance at 280 nm. The imidazole gradient is shown by the dashed black line. (b) SDS-PAGE analysis of the purified material (pooled peak fractions from panel ‘a’, lane 1) visualised by Coomassie blue staining. Protein size markers were run in lane ‘M’; their sizes (kDa) are shown on the left. This gel was run with just these two lanes and only these were scanned after staining. (c) DLS analysis of purified tetravalent mVLPs. The inset table shows the values of the DLS parameters. (d) EM picture of the VLPs after negative staining with uranyl acetate. The scale is shown at the top. (e–h) Dot blot assay to determine the relative proportion of E1, E2, E3 and E4 in T-mVLPs (details in Supplementary Protocol S3). Monovalent E VLPs corresponding to DENV serotypes 1 (E1), 2 (E2), 3 (E3) and 4 (E4) and T-mVLPs were spotted onto nitrocellulose strips and probed with anti-E mAbs specific to DENV-1 (mAb E24, panel ‘e’), DENV-2 (mAb 3H5, panel ‘f’), DENV-3 (mAb E1, panel ‘g’) and DENV-4 (mAb E42, panel ‘h’). T-mVLPs were spotted at two doses, the first (Tx) equivalent to the same dose as the monovalent E VLPs and the second (T4x) at four times the first dose. The histograms indicate the intensity of the dots in the blot (shown above; raw blots are shown on page 5 of the supplementary file) scanned using ImageJ software. Reactivities of the mAbs to the monovalent E1, E2, E3 and E4 VLPs are indicated by magenta, green, blue and black bars, respectively, in the histograms; mAb reactivities to the lower (Tx) and four-fold higher (T4x) doses of Tm-VLPs are indicated by the dark and light orange histogram bars, respectively. T-mVLP signal in the case of each serotype-specific mAb was equivalent to that of the corresponding monovalent E VLP when it was at 4x concentration indicating that it contains all four E proteins at 1:1:1:1 ratio.
