Figure 2
Production and characterization of the DIII-C-AP205 conjugate vaccine. A Derivatized AP205 (dAP205), DIII-C, and the dialysed conjugate vaccine (DIII-C-AP205) were analyzed by reducing, denaturing SDS-PAGE (left panel). Corresponding amounts of derivatized AP205, DIII-C, and of the non-dialysed (nd) conjugate vaccine were also analysed by non-reducing, non-denaturing SDS-PAGE (right panel). For identification of the coupling bands, proteins were separated on reducing, denaturing SDS-PAGE, blotted on nitrocellulose and detected with AP205- and His-tag- specific antibodies (middle panels). Bands corresponding to AP205-crosslinked DIII-C are indicated by arrows. The 27 kDa band which is visible in the SDS-PAGE and in the His-tag-specific Western Blot corresponds to dimeric DIII-C (*). B Size exclusion chromatography. A superdex 75 column was calibrated with a molecular weight (MW) calibration kit and then loaded sequentially with the indicated proteins. AP205 VLPs and the conjugate vaccine DIII-C-AP205 elute in the void volume of the column (V0, 40 ml) while purified DIII-C elutes at 72.4 ml.