Figure 1. CuMVTT-VLPs constitute an efficient platform for genetically fusing the receptor-binding domain (RBM)
A, schematic depiction of E. coli cells containing pETDuet-1-derived plasmid, allowing the coexpression of CuMVTT-RBM fusion and unmodified CMVTTgenes and production of mosaic VLPs termed (mCuMVTT-RBM), B, SDS-PAGE analysis of the production and sucrose gradient purification of mCuMVTT-RBM. M – protein size marker (Thermo Scientific), 0 – total proteins inE .coli C2566 cells before IPTG induction; T - total proteins inE .coli C2566 cells after IPTG induction and 18h cultivation at 20oC ; S – soluble proteins in E .coli C2566 cells after induction and 18 h cultivation; P – insoluble proteins inE .coli C2566 cells after induction and cultivation; 60 – 0 – sucrose gradient fractions after separation of cell lysate in Beckman SW32 rotor; mCuMVTT-RBM VLP containing sucrose fractions are labeled with red circle; C, native agarose gel analysis of the sucrose gradient fractions after sucrose gradient purification of mCuMVTT-RBM. M – DNA size marker (Thermo Scientific), 60–0– sucrose gradient fractions after separation of the cell lysate in Beckman SW32 rotor; mCuMVTT-RBM VLP containing sucrose fractions are labeled with red circle; D, SDS-PAGE analysis of purified mCuMVTT-RBM VLPs. M – protein size marker (Thermo Scientific), asterisk (blue) refers to unmodified CuMVTT monomer and asterisk (green) refers to genetically modified CuMVTT-RBM E, Electron microscopy analysis of purified mCuMVTT-RBM VLPs; F, Dynamic light scattering analysis of purified mCuMVTT-RBM VLPs. G, ACE2 binding to mCuMVTT-RBD vaccine candidate, CuMVTT and RBD alone were used as controls. Plates coated with 1µg/ml of ACE2. Binding was revealed using anti-CuMV mAb. One representative of 2 similar experiments is shown.