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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

Truncated psaK 5′ UTRs provide high levels of transgene production. Leaves of N. benthamiana were agroinfiltrated with non-replicating vectors containing different psaK 5′ UTRs, or the TMV 5′ UTR, upstream from the GFP gene (pGFPe-XX, where XX denotes the 5′ UTR). Agroinfiltrated leaf tissue was harvested at 5 DPI and leaf extracts were analyzed by SDS-PAGE followed by coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant protein bands as a loading control. Columns represent means ± standard error from four independently infiltrated samples. Two stars (∗∗) indicate p < 0.05 and three stars (∗∗∗) indicate p < 0.01 as compared to TMV by Student’s t-test.
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Figure 4: Truncated psaK 5′ UTRs provide high levels of transgene production. Leaves of N. benthamiana were agroinfiltrated with non-replicating vectors containing different psaK 5′ UTRs, or the TMV 5′ UTR, upstream from the GFP gene (pGFPe-XX, where XX denotes the 5′ UTR). Agroinfiltrated leaf tissue was harvested at 5 DPI and leaf extracts were analyzed by SDS-PAGE followed by coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant protein bands as a loading control. Columns represent means ± standard error from four independently infiltrated samples. Two stars (∗∗) indicate p < 0.05 and three stars (∗∗∗) indicate p < 0.01 as compared to TMV by Student’s t-test.

Mentions: We also wished to test the activity of plant-derived 5′ UTRs in the BeYDV system. It has been reported that a 5′ UTR derived from 63 nucleotides upstream from the start codon of the A. thaliana photosystem K subunit (AtPsaK) enhanced transgene expression in transgenic tobacco leaves (Agarwal et al., 2014). Using our system, we found that the 63nt AtPsaK 5′ UTR produced intense green fluorescence, and gel quantification data indicate that GFP production was increased by >20% compared to the TMV 5′ UTR (Figures 3A,B). The AtPsaK 5′ UTR (accession NM_102775) appears to be a truncation of the full-length 129 nt 5′ UTR. To further delineate the active region of the AtPsaK 5′ UTR, we created deletions at its 5′ and 3′ ends and tested their potential to enhance GFP production in non-replicating transient expression vectors. Using gel quantification, we found that a truncation removing nucleotides -1 to -23 upstream from the start codon resulted in a ∼14% decrease in GFP production, while a truncation removing nucleotides -42 to -63 resulted in a ∼13% increase in GFP production (Figure 4). A similar ∼12% enhancement was observed in replicating GFP vectors (Figure 3B).


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)

Truncated psaK 5′ UTRs provide high levels of transgene production. Leaves of N. benthamiana were agroinfiltrated with non-replicating vectors containing different psaK 5′ UTRs, or the TMV 5′ UTR, upstream from the GFP gene (pGFPe-XX, where XX denotes the 5′ UTR). Agroinfiltrated leaf tissue was harvested at 5 DPI and leaf extracts were analyzed by SDS-PAGE followed by coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant protein bands as a loading control. Columns represent means ± standard error from four independently infiltrated samples. Two stars (∗∗) indicate p < 0.05 and three stars (∗∗∗) indicate p < 0.01 as compared to TMV by Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4764687&req=5

Figure 4: Truncated psaK 5′ UTRs provide high levels of transgene production. Leaves of N. benthamiana were agroinfiltrated with non-replicating vectors containing different psaK 5′ UTRs, or the TMV 5′ UTR, upstream from the GFP gene (pGFPe-XX, where XX denotes the 5′ UTR). Agroinfiltrated leaf tissue was harvested at 5 DPI and leaf extracts were analyzed by SDS-PAGE followed by coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant protein bands as a loading control. Columns represent means ± standard error from four independently infiltrated samples. Two stars (∗∗) indicate p < 0.05 and three stars (∗∗∗) indicate p < 0.01 as compared to TMV by Student’s t-test.
Mentions: We also wished to test the activity of plant-derived 5′ UTRs in the BeYDV system. It has been reported that a 5′ UTR derived from 63 nucleotides upstream from the start codon of the A. thaliana photosystem K subunit (AtPsaK) enhanced transgene expression in transgenic tobacco leaves (Agarwal et al., 2014). Using our system, we found that the 63nt AtPsaK 5′ UTR produced intense green fluorescence, and gel quantification data indicate that GFP production was increased by >20% compared to the TMV 5′ UTR (Figures 3A,B). The AtPsaK 5′ UTR (accession NM_102775) appears to be a truncation of the full-length 129 nt 5′ UTR. To further delineate the active region of the AtPsaK 5′ UTR, we created deletions at its 5′ and 3′ ends and tested their potential to enhance GFP production in non-replicating transient expression vectors. Using gel quantification, we found that a truncation removing nucleotides -1 to -23 upstream from the start codon resulted in a ∼14% decrease in GFP production, while a truncation removing nucleotides -42 to -63 resulted in a ∼13% increase in GFP production (Figure 4). A similar ∼12% enhancement was observed in replicating GFP vectors (Figure 3B).

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