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.
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Abbreviations
- Ad:
-
Adenovirus
- Ad3:
-
Adenovirus serotype 3
- BSA:
-
Bovine serum albumin
- CBB:
-
Coomassie Brilliant Blue
- Dd:
-
Dodecahedron
- EM:
-
Electron microscopy
- FBS:
-
Fetal bovine serum
- FT:
-
Flow-through
- HRP:
-
Horseradish peroxidase
- IEC:
-
Ion exchange chromatography
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- MW:
-
Molecular weight
- Pb:
-
Penton base protein
- PBS:
-
Phosphate buffered saline
- SDS-PAGE:
-
SDS polyacrylamide gel electrophoresis
- SEC:
-
Size exclusion chromatography
- TMB:
-
3,3′,5,5′-Tetramethylbenzidine
- VLP:
-
Virus-like particle
- WB:
-
Western blot
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Acknowledgments
This study was supported by Polish National Science Center (DEC-2013/09/B/NZ3/02327). The electron microscope facility used in this work is supported by the Rhône-Alpes Region, FRM, FEDER of CNRS, CEA, University of Grenoble, EMBL, and the GIS-Infrastructures of IBISA. We are grateful to Irina Kikhno (Institute of Molecular Biology & Genetics of Ukrainian Academy of Science, Kiev, Ukraine) for the plasmid for construction of the E. coli expression vector and to Elzbieta Grzesiuk’s team for their collaboration in protein purification.
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I. Szurgot and M. Jedynak contributed equally to this work.
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Szurgot, I., Jedynak, M., Podsiadla-Bialoskorska, M. et al. Adenovirus Dodecahedron, a VLP, Can be Purified by Size Exclusion Chromatography Instead of Time-Consuming Sucrose Density Gradient Centrifugation. Mol Biotechnol 57, 565–573 (2015). https://doi.org/10.1007/s12033-015-9850-9
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DOI: https://doi.org/10.1007/s12033-015-9850-9