Dataset Information

Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in Nicotiana benthamiana Leaves.

ABSTRACT: Plants are a promising platform to produce biopharmaceutical proteins, however, the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and RepA strongly affects the efficiency of replication. Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death. We find that a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation. A single nucleotide change in the 5' untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies. We also find that replicating vectors achieve optimal expression with lower Agrobacterium concentrations than non-replicating vectors, further reducing cell death. Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5' UTR does not.

SUBMITTER: Diamos AG

PROVIDER: S-EPMC6333858 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in <i>Nicotiana benthamiana</i> Leaves.

Diamos Andrew G AG Mason Hugh S HS

Frontiers in plant science 20190109

Plants are a promising platform to produce biopharmaceutical proteins, however, the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and Rep ...[more]

PMID: 30687368

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Project description:We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5' untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5' UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5' UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.

Dataset Information

Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in Nicotiana benthamiana Leaves.

Publications

Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in <i>Nicotiana benthamiana</i> Leaves.

Similar Datasets

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