5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

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.

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

“…In 2016, the BeYDV vector was optimized by modifying its 5′ and 3′ untranslated regions. Use of the optimized vector significantly enhanced the yield of recombinant protein (1.8 mg/L) in Nicotiana benthamiana leaves, which represented the highest yields of ZMapp ever reported [72]. Wang and Lauren (2014) successfully expressed NK in tobacco leaves utilizing the BeYDV system [73].…”

Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity

“…Plant expression systems are highly scalable, do not contain animal pathogens, and also avoid many of the costs of traditional expression systems, such as expensive bioreactors, thereby allowing cheaper production of biological products [38][39][40]. We have previously described a robust plant expression system based on the bean yellow dwarf virus that permits high levels of protein production in N. benthamiana [35,[41][42][43]. A recent study with plastidengineered tobacco plants further demonstrated the viability of plant-based protein production in a field setting.…”

“…The resulting constructs (Fig. 1A) were placed in replicating geminiviral plant expression vectors [35], agroinfiltrated into N. benthamiana leaves, and monitored for GFP expression. GFP N-RICs produced bright green fluorescence, while GFP C-RICs produced lower levels of GFP fluorescence ( Fig.…”

Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans

“…It is cheap, fast and can be easily up-scaled. A production yield of 1 mg of rituximab antibody per gram leaf (fresh weight) has been reported (Diamos et al, 2016). However, down-stream processing (e.g., purification) represents an important part of the production costs which should be lower with suspension cells when the protein of interest is secreted.…”