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.