5′ and 3′ untranslated regions strongly enhance performance of Geminiviral replicons in Nicotiana benthamiana leaves

Andrew G. Diamos, Sun H. Rosenthal, Hugh Mason

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article number200
JournalFrontiers in Plant Science
Volume7
Issue numberFEB2016
DOIs
StatePublished - Feb 24 2016

Fingerprint

replicon
Nicotiana benthamiana
5' untranslated regions
3' untranslated regions
Bean yellow dwarf virus
Tobacco mosaic virus
leaves
recombinant proteins
proteins
cell death
monoclonal antibodies
tobacco
Norwalk virus
extensin
Geminiviridae
photosystem I
coat proteins
Agrobacterium
transgenes
production technology

Keywords

  • 3′ untranslated regions
  • 5′ untranslated regions
  • Geminivirus
  • Monoclonal antibody
  • Nicotiana benthamiana
  • Transient expression
  • Virus-like particle

ASJC Scopus subject areas

  • Plant Science

Cite this

5′ and 3′ untranslated regions strongly enhance performance of Geminiviral replicons in Nicotiana benthamiana leaves. / Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh.

In: Frontiers in Plant Science, Vol. 7, No. FEB2016, 200, 24.02.2016.

Research output: Contribution to journalArticle

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abstract = "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.",
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