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Enhancement of plant expression vectors using replication and silencing suppressor elements
Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used...
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2019
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| _version_ | 1867613169012178944 |
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| access_status_str | Open Access |
| author | Jacobs, Raygaana |
| author2 | Hitzeroth, Inga |
| author_browse | Hitzeroth, Inga Jacobs, Raygaana |
| author_facet | Hitzeroth, Inga Jacobs, Raygaana |
| author_sort | Jacobs, Raygaana |
| collection | Thesis |
| description | Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/29414 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:31:52.071Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/29414 Enhancement of plant expression vectors using replication and silencing suppressor elements Jacobs, Raygaana Hitzeroth, Inga Rybicki, Edward Regnard, Guy Molecular and Cell Biology Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines. 2019-02-08T09:06:55Z 2019-02-08T09:06:55Z 2018 2019-02-08T08:45:09Z Master Thesis Masters MSc http://hdl.handle.net/11427/29414 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Molecular and Cell Biology Jacobs, Raygaana Enhancement of plant expression vectors using replication and silencing suppressor elements |
| thesis_degree_str | Master's |
| title | Enhancement of plant expression vectors using replication and silencing suppressor elements |
| title_full | Enhancement of plant expression vectors using replication and silencing suppressor elements |
| title_fullStr | Enhancement of plant expression vectors using replication and silencing suppressor elements |
| title_full_unstemmed | Enhancement of plant expression vectors using replication and silencing suppressor elements |
| title_short | Enhancement of plant expression vectors using replication and silencing suppressor elements |
| title_sort | enhancement of plant expression vectors using replication and silencing suppressor elements |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/29414 |
| work_keys_str_mv | AT jacobsraygaana enhancementofplantexpressionvectorsusingreplicationandsilencingsuppressorelements |