Recombinant Nicotiana tabacum 47 kDa cell wall protein
Description
Identification and Characterization
The 47 kDa cell wall protein in N. tabacum has been associated with both native and recombinant forms:
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Native Protein: A cell wall invertase inhibitor (INH) was purified from tobacco cells, encoded by the Nt-inh1 gene. This protein binds specifically to cell wall invertase (CWI) to regulate sucrose metabolism during stress or developmental transitions .
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Recombinant Variants: Transgenic tobacco lines have been engineered to express heterologous 47 kDa proteins, such as the Mycobacterium tuberculosis APA antigen. This recombinant APA migrates at 45/47 kDa on SDS-PAGE due to glycosylation and proline-rich sequences, despite a theoretical molecular weight of ~28 kDa .
| Protein Type | Origin | Molecular Weight (Observed) | Function | Reference |
|---|---|---|---|---|
| Native INH | N. tabacum | ~47 kDa | CWI inhibition, sucrose signaling | |
| Recombinant APA | M. tuberculosis | 45/47 kDa | Immunogenic antigen production |
Transient Expression
N. benthamiana (a close relative of N. tabacum) is widely used for transient expression due to:
Example Protocol:
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Codon optimization of target genes (e.g., fungal effectors or immune receptors) for tobacco expression .
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Fusion with signal peptides (e.g., α-coixin) for endoplasmic reticulum targeting and glycosylation .
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Affinity purification using Twin-Strep-tag® systems, which tolerate reducing agents like DTT .
Stable Transformation
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Seed-specific promoters (e.g., γ-kafirin from sorghum) direct recombinant protein accumulation in tobacco seeds, achieving yields up to 1.5% of total soluble protein (TSP) .
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Subcellular targeting to protein storage vacuoles enhances stability and simplifies extraction .
Glycosylation and Post-Translational Modifications
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Recombinant APA produced in tobacco seeds exhibits glycosylation patterns distinct from bacterial forms, confirmed by Concanavalin A binding and 2D electrophoresis .
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Nt-inh1 lacks predicted N-glycosylation sites but contains conserved cysteine residues critical for inhibitory activity .
Kinase Activity
Wall-associated kinases (WAKs) in N. tabacum, such as NtWAK11 and NtWAK32, are plasma membrane-localized proteins with phosphorylation activity. Their extracellular domains interact with the cell wall, mediating stress signaling .
Biomedical Research
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Vaccine Production: Recombinant APA from tobacco seeds serves as a candidate for tuberculosis vaccines, leveraging plant-specific glycosylation for enhanced immunogenicity .
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Antibody Synthesis: Cell wall-targeted monoclonal antibodies (e.g., mAb 14D9) achieve yields up to 1.73% TSP in tobacco .
Agricultural Biotechnology
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Stress Resistance: WAK proteins regulate responses to NaCl, PEG, and ABA treatments, making them targets for engineering drought-tolerant crops .
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Pathogen Defense: Recombinant NLR immune receptors (e.g., ROQ1 resistosome) are purified from tobacco for structural studies on plant-pathogen interactions .
Challenges and Innovations
Product Specs
Target Background
Q&A
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What experimental designs are effective for studying the function of the 47 kDa cell wall protein in Nicotiana tabacum?
To investigate the function of the recombinant 47 kDa cell wall protein, researchers can employ a combination of molecular biology techniques and proteomic analyses. An effective experimental design would include:
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Gene Cloning and Transformation: Utilize Agrobacterium-mediated transformation to introduce the recombinant gene into Nicotiana tabacum plants, allowing for stable expression of the protein.
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Functional Characterization: Conduct loss-of-function studies using RNA interference or CRISPR-Cas9 to assess phenotypic changes in plants lacking the 47 kDa protein. Comparative analyses between wild-type and mutant plants can reveal insights into its role in cell wall integrity and plant defense mechanisms.
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Proteomic Analysis: Utilize two-dimensional electrophoresis followed by mass spectrometry to analyze changes in the proteome upon expression of the recombinant protein. This can help identify interacting partners and downstream signaling pathways affected by the 47 kDa protein .
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How can data contradictions in studies involving recombinant Nicotiana tabacum proteins be analyzed?
Data contradictions in studies involving recombinant proteins can be analyzed through:
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Reproducibility Checks: Conduct independent experiments to verify initial findings. This includes repeating assays under varying conditions (e.g., different growth media or stress treatments) to determine if results are consistent.
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Statistical Analysis: Employ robust statistical methods to analyze data sets, focusing on variance and significance levels. Techniques like ANOVA can help discern whether observed differences are statistically significant or due to experimental error.
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Integration of Multi-Omics Data: Combine transcriptomic, proteomic, and metabolomic data to provide a holistic view of how the recombinant protein influences cellular processes. This approach can help reconcile discrepancies by identifying broader biological contexts .
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What advanced techniques can be used to quantify the expression levels of the 47 kDa cell wall protein in Nicotiana tabacum?
Advanced techniques for quantifying expression levels include:
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Quantitative PCR (qPCR): This method allows for precise quantification of mRNA levels associated with the 47 kDa protein. Specific primers can be designed to amplify target sequences, providing insights into transcriptional regulation.
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Western Blotting: Following protein extraction, Western blotting can be employed using antibodies specific to the 47 kDa protein. This technique enables direct measurement of protein abundance and can be coupled with densitometry for quantitative analysis.
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Mass Spectrometry-Based Proteomics: Utilize label-free quantitation or isobaric tagging methods (like iTRAQ) to measure relative abundance of the 47 kDa protein across different samples or treatments, allowing for a comprehensive understanding of its expression dynamics .
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What are the implications of post-translational modifications on the functionality of recombinant Nicotiana tabacum proteins?
Post-translational modifications (PTMs) significantly influence protein functionality in several ways:
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Activity Regulation: Modifications such as phosphorylation or glycosylation can alter enzymatic activity, stability, and interaction with other cellular molecules. For example, glycosylation may affect cell wall localization and interactions with other cell wall components.
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Protein Stability: PTMs can enhance or reduce protein stability, impacting its half-life within cells. Understanding these modifications is critical for optimizing conditions for recombinant protein expression.
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Functional Assays: To assess the impact of PTMs on functionality, researchers should conduct assays that mimic physiological conditions (e.g., specific pH or ionic strength) under which these modifications occur naturally. This will provide insights into how PTMs affect biological activity .
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How does environmental stress affect the expression and function of recombinant Nicotiana tabacum cell wall proteins?
Environmental stressors such as drought, salinity, or pathogen attack can significantly alter the expression and function of cell wall proteins:
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Gene Expression Profiling: Use RNA sequencing or qPCR to evaluate changes in mRNA levels of the 47 kDa protein under various stress conditions. This profiling helps identify stress-responsive regulatory elements within its promoter region.
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Functional Analysis Under Stress: Conduct functional assays to determine how stress affects the structural integrity and biochemical properties of the cell wall. For instance, analyzing cell wall composition before and after stress exposure can reveal adaptive mechanisms.
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Proteomic Shifts: Investigate shifts in proteomic profiles using mass spectrometry to identify other proteins co-regulated with the 47 kDa protein during stress responses, providing insights into potential pathways involved in plant resilience .
