Recombinant Nicotiana tabacum 70 kDa cell wall protein

What is the optimal expression system for recombinant proteins in Nicotiana tabacum?

Agrobacterium-mediated transient expression systems, particularly syringe agroinfiltration into tobacco leaves, offer an efficient and rapid approach for recombinant protein production. This method provides a convenient way to produce recombinant proteins with greater expression throughout the plant leaf. For human tropoelastin protein expression, Agrobacterium-mediated transient expression via agroinfiltration has demonstrated successful results with detectable expression starting from 48 hours post-infiltration and persisting for up to 10 days . The system allows researchers to produce recombinant proteins in a significantly reduced timeframe compared to stable transformation methods.

How can successful transformation and expression of recombinant proteins in Nicotiana tabacum be confirmed?

A multi-level confirmation approach is recommended:

• DNA level: Southern blot analysis to confirm gene integration into plant genome

• RNA level: RT-PCR to verify transcription using gene-specific primers

• Protein level: ELISA and Western blot analysis using specific antibodies

For example, in tropoelastin expression studies, Southern blot analysis detected a 2175 bp fragment representing the ELN orf, RT-PCR amplified an ~800 bp fragment confirming transcription, and Western blot analysis revealed a parent band at ~70 kDa for freshly extracted protein . This comprehensive verification ensures confirmation at each stage of expression.

What is the optimal harvest time for recombinant proteins from agroinfiltrated Nicotiana tabacum?

Based on tropoelastin expression research, protein levels peak at the 5th day post-infiltration (d.p.i.), followed by a gradual decrease at days 7 and 10. This was confirmed through both ELISA and Western blot analyses . The temporal expression pattern can be visualized in the following data from ELISA readings:

Therefore, researchers are recommended to harvest and purify recombinant proteins from infiltrated leaves at the 5th day post-infiltration to obtain maximum yield .

What strategies can mitigate proteolytic degradation of recombinant proteins in Nicotiana tabacum extracts?

Protein degradation is a significant challenge in plant expression systems, as evidenced by tropoelastin degradation patterns. Fresh extracts showed a single band at ~70 kDa, while stored samples displayed additional degraded bands at ~55 and ~45 kDa . This degradation is likely due to proteolytic activity in tobacco tissues, which contain over 90% water, potentially enhancing proteolysis.

Effective strategies include:

• Immediate processing of fresh leaf material

• Addition of protease inhibitor cocktails to extraction buffers

• Optimization of extraction conditions (temperature, pH)

• Codon optimization to enhance protein stability (as demonstrated by improving CAI from 0.70 to 0.88 for tropoelastin)

• Addition of stabilizing agents such as polyvinylpyrrolidone, which enhanced rhEPO stability by 5.6-fold in spent medium

How does subcellular targeting affect recombinant protein yield and stability in Nicotiana tabacum?

Subcellular targeting significantly impacts both yield and stability of recombinant proteins. In the rhEPO study, the presence of the endoplasmic reticulum (ER) signal peptide (calreticulin) was essential for protein secretion into the spent medium; no protein was detected from hairy root cultures without the ER signal peptide .

Different compartments offer various advantages:

• ER retention: Often increases yield through reduced proteolytic degradation

• Secretory pathway: Enables protein collection from spent medium

• Specialized compartments: May offer protection from proteases

For example, researchers investigating sieve element-specific proteins in tobacco found that targeting to specialized ER segments using reticulon-like proteins provided compartmentalization that could protect proteins from degradation .

How can protein extraction protocols be optimized to maintain structural integrity of large recombinant proteins from Nicotiana tabacum?

For large proteins like the 70 kDa tropoelastin, maintaining structural integrity during extraction is crucial. The following protocol has proven effective:

• Harvest and immediately process fresh leaf material (avoid freezing when possible)

• Grind tissues in liquid nitrogen to prevent thermal degradation

• Extract using SDS-extraction buffer (2% SDS, 0.1% bromophenol blue, 10% glycerol)

• Clarify extracts by centrifugation at 14,000 g for 20 min at 4°C

• Transfer supernatant to fresh tubes and determine protein content via Bradford assay

• Process immediately for analysis or purification

When protein storage is necessary, add appropriate protease inhibitors and maintain samples at -80°C, but expect some degradation as observed with tropoelastin samples .

What protein detection methods offer highest sensitivity for low-abundance recombinant proteins in Nicotiana tabacum?

Multiple complementary detection methods are recommended, each with specific advantages:

• Direct ELISA: Provides quantitative measurement with high sensitivity. For tropoelastin detection, ELISA could detect expression differences between time points and was able to monitor expression kinetics effectively .

• Western Blot: Enables visualization of the target protein and any degradation products or isoforms. For tropoelastin, it effectively detected the parent 70 kDa band and degradation products at ~55 and ~45 kDa. The protocol involves:

  • SDS-PAGE separation

  • Transfer to PVDF membrane

  • Blocking for at least 2 hours

  • Probing with primary antibody (e.g., anti-tropoelastin at 1:500 dilution)

  • Incubation with alkaline phosphatase-conjugated secondary antibody (1:5000)

  • Development with BCIP/NBT

• Fluorescence Microscopy: For fluorescently tagged proteins, confocal microscopy offers highest sensitivity, detecting proteins even when expression levels are too low for standard epifluorescence microscopes .

How can secretion of recombinant proteins from Nicotiana tabacum be enhanced?

Based on the rhEPO study, several strategies can enhance protein secretion:

• Addition of ER signal peptides (e.g., calreticulin) to direct proteins to the secretory pathway

• Addition of polyvinylpyrrolidone to the medium to stabilize secreted proteins

• Optimization of culture conditions (temperature, light, medium composition)

The addition of polyvinylpyrrolidone enhanced rhEPO stabilization, leading to a 5.6-fold increase in concentration to a maximum of 185.48 pg rhEPO per gram fresh weight of hairy root cultures .

What considerations are important when designing codon optimization strategies for mammalian proteins in Nicotiana tabacum?

Codon optimization is crucial for efficient expression of mammalian genes in plant systems. For tropoelastin expression, researchers improved the codon adaptation index (CAI) from 0.70 to 0.88, which enhanced expression without altering the protein's primary structure .

Key considerations include:

• Analysis of N. tabacum codon usage preferences

• Adjustment of rare codons while maintaining the amino acid sequence

• Removal of cryptic splice sites and unwanted regulatory elements

• Optimization of GC content

• Elimination of sequence repetitions that might cause recombination

What controls should be included when evaluating recombinant protein expression in Nicotiana tabacum?

A comprehensive set of controls ensures reliable interpretation of results:

• Negative controls:

  • Uninfiltrated plant leaves

  • Plants infiltrated with Agrobacterium carrying empty vector

• Positive controls:

  • Purified recombinant protein (if available)

  • Digested recombinant vector (e.g., pUC57-ELN for Southern blot)

• Time-course controls:

  • Samples collected at multiple time points (e.g., 3, 5, 7, and 10 days post-infiltration)

  • Different dilutions of protein extract to ensure detection within linear range

These controls help distinguish true expression from background signals and determine optimal expression conditions.

How can biological activity of plant-produced recombinant proteins be accurately assessed?

Functional assessment of recombinant proteins is essential to confirm their biological equivalence to native proteins:

• Protein-specific functional assays:

  • For rhEPO, cell proliferation and survival parameters were tested in retinal pigment epithelial cells (ARPE)

  • For structural proteins like tropoelastin, physical properties testing may be appropriate

• Comparative analysis:

  • Direct comparison with the same protein produced in conventional systems

  • Analysis of post-translational modifications

  • Structural characterization (circular dichroism, mass spectrometry)

• Purification considerations:

  • HPLC purification to isolate intact protein

  • Removal of plant-specific compounds that might interfere with activity assays

How should experiments be designed to study protein degradation patterns in Nicotiana tabacum extracts?

Based on the observed degradation patterns of tropoelastin (from 70 kDa to 55 and 45 kDa bands), a systematic approach includes:

• Time-course experiments:

  • Process identical samples at different time points after extraction

  • Compare fresh extracts vs. stored extracts (different storage conditions)

• Inhibitor studies:

  • Test various protease inhibitor cocktails

  • Evaluate single inhibitors to identify specific proteases involved

• Extraction condition variations:

  • Test different buffers, pH conditions, temperatures

  • Compare mechanical disruption methods

• Analysis methods:

  • Western blot to visualize degradation patterns

  • Mass spectrometry to identify cleavage sites

  • N-terminal sequencing of degradation products

Why might multiple bands appear on Western blots of recombinant proteins from Nicotiana tabacum?

Multiple bands on Western blots can result from several factors:

• Proteolytic degradation: As observed with tropoelastin, where fresh extracts showed one band at ~70 kDa, while stored samples displayed additional bands at ~55 and ~45 kDa

• Post-translational modifications:

  • Differential glycosylation

  • Phosphorylation states

  • Other modifications specific to plant expression systems

• Technical issues:

  • Incomplete denaturation prior to SDS-PAGE

  • Non-specific antibody binding

  • Sample overloading

• Alternative splicing or translation initiation sites

To distinguish between these possibilities, researchers should compare fresh and stored samples, use different extraction methods, and consider mass spectrometry analysis to identify the nature of additional bands.

What factors influence yield variability in Agrobacterium-mediated transient expression systems?

Several factors affect recombinant protein yield in tobacco agroinfiltration systems:

• Temporal factors:

  • Time post-infiltration (optimal was 5 days for tropoelastin)

  • Plant age and physiological state

• Biological factors:

  • Agrobacterium strain and optical density during infiltration

  • Leaf position and developmental stage

  • Plant growing conditions (light, temperature, nutrition)

• Technical factors:

  • Infiltration technique (complete vs. partial leaf coverage)

  • Codon optimization (CAI improvement was important)

  • Vector design (promoter strength, enhancers)

• Biochemical factors:

  • Protein stability in plant tissue

  • Proteolysis due to high water content (>90% in tobacco tissues)

  • Inherent protein characteristics

Controlling these variables through standardized protocols can significantly reduce yield variability between experiments.

How can contradictory results between different protein detection methods be resolved?

When detection methods yield contradictory results (e.g., positive ELISA but negative Western blot), consider:

• Method sensitivity differences:

  • ELISA typically has lower detection limits than Western blot

  • RT-PCR detects mRNA but doesn't confirm protein translation

• Protein conformation effects:

  • Native vs. denatured protein detection

  • Epitope accessibility in different assays

• Systematic approach to resolution:

  • Increase sample concentration for less sensitive methods

  • Use alternative antibodies that recognize different epitopes

  • Employ purification steps before analysis

  • Validate with orthogonal methods (mass spectrometry)

  • Consider time-course analysis (protein might be expressed transiently)

When tropoelastin expression was studied, researchers used multiple detection methods (Southern blot, RT-PCR, ELISA, and Western blot) to provide comprehensive confirmation across the central dogma of molecular biology .