Abstract
Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Agrobacterium tumefaciens / genetics
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Allergens / biosynthesis
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Allergens / genetics
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Analysis of Variance
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Enzyme-Linked Immunosorbent Assay / standards
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Gene Expression*
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Genetic Vectors
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Luminescent Proteins / biosynthesis
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Luminescent Proteins / genetics
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Nicotiana / genetics*
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Nicotiana / metabolism
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Organ Specificity
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Plant Leaves / genetics*
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Plant Leaves / metabolism
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Plant Proteins / biosynthesis
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Plant Proteins / genetics
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Plants, Genetically Modified / genetics*
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Plants, Genetically Modified / metabolism
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Protein Stability
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / genetics
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Reference Standards
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Single-Chain Antibodies / biosynthesis
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Single-Chain Antibodies / genetics
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Transcriptome
Substances
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Allergens
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Luminescent Proteins
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Plant Proteins
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Recombinant Proteins
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Single-Chain Antibodies
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fluorescent protein 583
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PHLPI protein, Phleum pratense
Grants and funding
This work was supported by the founding initiative “Transnational PLant Alliance for Novel Technologies - towards implementing the Knowledge-Based Bio-Economy in Europe” (PLANT-KBBE, FKZ0315456B) of the German Federal Ministry of Education and Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.