Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system

• Published in: Applied microbiology and biotechnology 2014-10, Vol.98 (19), p.8281-8290
• Main Authors: Moon, Ki-Beom, Lee, Jisu, Kang, Sebyung, Kim, Moonil, Mason, Hugh S, Jeon, Jae-Heung, Kim, Hyun-Soon
• Format: Article
• Language: English
• Subjects: agroinfiltration; Applied Genetics and Molecular Biotechnology; Biomedical and Life Sciences; Biotechnology; Brome mosaic virus; Bromovirus - genetics; Bromovirus - metabolism; Capsid Proteins - genetics; Capsid Proteins - metabolism; Cloning; coat proteins; Cucumber mosaic virus; Cucumovirus - genetics; Cucumovirus - metabolism; Deoxyribonucleic acid; DNA; Electrons; Engineering research; Enzymes; Gene Expression; gene overexpression; Genetic aspects; Genetic Vectors - genetics; Genetic Vectors - metabolism; Genomes; Growth conditions; High temperature; Humidity; Leaves; Life Sciences; Maize rayado fino virus; Materials science; medicine; Microbial Genetics and Genomics; Microbiology; Monoclonal antibodies; Nanomaterials; Nicotiana; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana benthamiana; Physiological aspects; Plant growth; polyacrylamide gel electrophoresis; Protein expression; protein synthesis; Proteins; quantitative analysis; Replicon; Research centers; Studies; sucrose; Systems engineering; Transgenic plants; Transmission electron microscopy; Tymoviridae - genetics; Tymoviridae - metabolism; ultracentrifugation; Vectors (Biology); Virion - genetics; Virion - metabolism; virus-like particles; Viruses
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-014-5901-6

Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g⁻¹ leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.