Adenovirus Dodecahedron, a VLP, Can be Purified by Size Exclusion Chromatography Instead of Time-Consuming Sucrose Density Gradient Centrifugation

Abstract

Adenoviral dodecahedron (Dd) is a virus-like particle composed of twelve pentameric penton base (Pb) proteins, responsible for adenovirus cell penetration. It is generated spontaneously in the baculovirus system upon expression of the Pb gene of adenovirus serotype 3. This particle shows remarkable cell penetration ability with 2,00,000-3,00,000 Dd internalized into one cell in culture, conceivably delivering several millions of foreign cargo molecules to the target cell. We have used it in the past for delivery of small drugs as well as a vaccination platform, in which Dd serves as a particulate vaccine delivery system. Since development of new biomedicals depends strongly on the cost of their expression and purification, we attempted, albeit unsuccessfully, to obtain Dd expression in bacteria. We therefore retained its expression in the baculovirus/insect cells system but introduced significant improvements in the protocols for Dd expression and purification, leading to considerable savings in time and improved yield.

Novel coronavirus-like particles targeting cells lining the respiratory tract

“…Our efforts to purify HCoV-NL63 VLPs using this method failed. Most of the alternative purification methods are based on size-exclusion column purification [60][61][62]. A limitation of this approach is that a relatively low amount of material can be loaded onto a size-exclusion column.…”

Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron

“…We proposed simple protocol for Dd lyophilization for prolonged storage. Electron microscopy and In-cell ELISA analyses of water-reconstructed samples indicated that Dd purified in two-step protocol based on size exclusion and anion exchange chromatography 18 can be frozen and lyophilized in the presence of sodium chloride and cryoprotectants (sucrose and mannitol), without inducing aggregation, losing structural integrity or penetration capabilities (Figure 8). Of note, even preparations lyophilized in the absence of cryoprotectants after reconstitution in water still contained a large proportion of well-formed dodecahedra, although a significant amount of free pentameric bases was observed (data not shown).…”

On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines

“…For Dd expression, Trichoplusia ni (HighFive) cells (2 × 10 6 cells/ml) grown in suspension were infected with the recombinant baculovirus at multiplicity of infection 4 and cultured for 48 h. Expressing cells were lysed and Dd was purified from the supernatant by size exclusion chromatography followed by anion exchange chromatography, as described previously [24]. Dd preparation was concentrated using Amicon Ultra concentrators (Merck Millipore), protein concentration was determined by NanoDrop ND-200 UV-Vis Spectrophotometer (Thermo Scientific).…”