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

Diamos AG, Rosenthal SH, Mason HS - Front Plant Sci (2016)

Bottom Line: 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 also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Vaccinology, Biodesign Institute, and School of Life Sciences, Arizona State University, Tempe AZ, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Extensin terminator increases transgene production and mRNA accumulation. (A) Fluorimetric analysis of GFP production. N. benthamiana leaves were agroinfiltrated with BeYDV vectors containing either the vspB terminator (gray bars, pBYGFP.R) or tobacco extensin terminator (black bars, pBYGFP.EFR). Leaves from agroinfiltrated tissue were harvested at 3–5 DPI and analyzed for GFP production by spectrofluorimetry using excitation and emission wavelengths of 485 and 538 nm. GFP production was normalized by total soluble protein. Columns represent means ± SD from six independently infiltrated samples. (B) Total RNA from agroinfiltrated N. benthamiana leaves were analyzed for GFP mRNA accumulation by quantitative RT-PCR at 3 DPI. Translation elongation factor 1α was used as an internal loading control. Columns represent means ± SD from four independent infiltrated samples. (C) NVCP production. Protein extracts from N. benthamiana leaves agroinfiltrated with either the vspB terminator (gray bars, pBYNVCP.R) or the tobacco extensin terminator (black bars, pBYNVCP.REF) harvested at 3 DPI were analyzed for NVCP production by ELISA and normalized by total soluble protein. Columns represent means ± SD for nine independently infiltrated samples.
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Figure 2: Extensin terminator increases transgene production and mRNA accumulation. (A) Fluorimetric analysis of GFP production. N. benthamiana leaves were agroinfiltrated with BeYDV vectors containing either the vspB terminator (gray bars, pBYGFP.R) or tobacco extensin terminator (black bars, pBYGFP.EFR). Leaves from agroinfiltrated tissue were harvested at 3–5 DPI and analyzed for GFP production by spectrofluorimetry using excitation and emission wavelengths of 485 and 538 nm. GFP production was normalized by total soluble protein. Columns represent means ± SD from six independently infiltrated samples. (B) Total RNA from agroinfiltrated N. benthamiana leaves were analyzed for GFP mRNA accumulation by quantitative RT-PCR at 3 DPI. Translation elongation factor 1α was used as an internal loading control. Columns represent means ± SD from four independent infiltrated samples. (C) NVCP production. Protein extracts from N. benthamiana leaves agroinfiltrated with either the vspB terminator (gray bars, pBYNVCP.R) or the tobacco extensin terminator (black bars, pBYNVCP.REF) harvested at 3 DPI were analyzed for NVCP production by ELISA and normalized by total soluble protein. Columns represent means ± SD for nine independently infiltrated samples.

Mentions: To test the potential of the tobacco extensin terminator to enhance protein production in the BeYDV system, N. benthamiana leaves were agroinfiltrated with replicating GFP vectors containing either the soybean vspB terminator or the tobacco extensin terminator (Figure 1). Protein extracts from agroinfiltrated leaf samples were analyzed from 3 to 5 DPI by spectrofluorimetry. Vectors containing the tobacco extensin terminator provided an approximately 2.5-fold increase in GFP production compared to vspB (Figure 2A). Quantitative real-time RT-PCR showed that the increase in GFP was associated with a similar 2.5-fold increase in GFP transcripts (Figure 2B).


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

Diamos AG, Rosenthal SH, Mason HS - Front Plant Sci (2016)

Extensin terminator increases transgene production and mRNA accumulation. (A) Fluorimetric analysis of GFP production. N. benthamiana leaves were agroinfiltrated with BeYDV vectors containing either the vspB terminator (gray bars, pBYGFP.R) or tobacco extensin terminator (black bars, pBYGFP.EFR). Leaves from agroinfiltrated tissue were harvested at 3–5 DPI and analyzed for GFP production by spectrofluorimetry using excitation and emission wavelengths of 485 and 538 nm. GFP production was normalized by total soluble protein. Columns represent means ± SD from six independently infiltrated samples. (B) Total RNA from agroinfiltrated N. benthamiana leaves were analyzed for GFP mRNA accumulation by quantitative RT-PCR at 3 DPI. Translation elongation factor 1α was used as an internal loading control. Columns represent means ± SD from four independent infiltrated samples. (C) NVCP production. Protein extracts from N. benthamiana leaves agroinfiltrated with either the vspB terminator (gray bars, pBYNVCP.R) or the tobacco extensin terminator (black bars, pBYNVCP.REF) harvested at 3 DPI were analyzed for NVCP production by ELISA and normalized by total soluble protein. Columns represent means ± SD for nine independently infiltrated samples.
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Figure 2: Extensin terminator increases transgene production and mRNA accumulation. (A) Fluorimetric analysis of GFP production. N. benthamiana leaves were agroinfiltrated with BeYDV vectors containing either the vspB terminator (gray bars, pBYGFP.R) or tobacco extensin terminator (black bars, pBYGFP.EFR). Leaves from agroinfiltrated tissue were harvested at 3–5 DPI and analyzed for GFP production by spectrofluorimetry using excitation and emission wavelengths of 485 and 538 nm. GFP production was normalized by total soluble protein. Columns represent means ± SD from six independently infiltrated samples. (B) Total RNA from agroinfiltrated N. benthamiana leaves were analyzed for GFP mRNA accumulation by quantitative RT-PCR at 3 DPI. Translation elongation factor 1α was used as an internal loading control. Columns represent means ± SD from four independent infiltrated samples. (C) NVCP production. Protein extracts from N. benthamiana leaves agroinfiltrated with either the vspB terminator (gray bars, pBYNVCP.R) or the tobacco extensin terminator (black bars, pBYNVCP.REF) harvested at 3 DPI were analyzed for NVCP production by ELISA and normalized by total soluble protein. Columns represent means ± SD for nine independently infiltrated samples.
Mentions: To test the potential of the tobacco extensin terminator to enhance protein production in the BeYDV system, N. benthamiana leaves were agroinfiltrated with replicating GFP vectors containing either the soybean vspB terminator or the tobacco extensin terminator (Figure 1). Protein extracts from agroinfiltrated leaf samples were analyzed from 3 to 5 DPI by spectrofluorimetry. Vectors containing the tobacco extensin terminator provided an approximately 2.5-fold increase in GFP production compared to vspB (Figure 2A). Quantitative real-time RT-PCR showed that the increase in GFP was associated with a similar 2.5-fold increase in GFP transcripts (Figure 2B).

Bottom Line: 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 also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Vaccinology, Biodesign Institute, and School of Life Sciences, Arizona State University, Tempe AZ, USA.

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.

No MeSH data available.


Related in: MedlinePlus