Recombinant Daucus carota 46 kDa cell wall protein

What is the Daucus carota 46 kDa cell wall protein and what is its function in plant tissues?

The Daucus carota 46 kDa cell wall protein is a structural glycoprotein component of carrot cell walls that plays crucial roles in maintaining cell wall integrity and participating in plant defense mechanisms. Similar to other characterized carrot cell wall proteins, it likely contains a significant proportion of proline-rich regions and undergoes post-translational modifications including hydroxylation and glycosylation . In native tissues, this protein contributes to the structural framework of the cell wall and may be involved in responses to environmental stresses, similar to other identified carrot defense proteins such as CR16 (Major allergen Dau c1) .

How does the 46 kDa cell wall protein compare structurally to other characterized Daucus carota cell wall proteins?

The 46 kDa cell wall protein shares structural similarities with other characterized carrot cell wall proteins, particularly the 55 kDa major cell wall glycoprotein identified in carrot disc studies . Research indicates that carrot cell wall proteins typically contain multiple proline-rich domains, as observed in the 14 kDa proline-rich protein DC2.15, which features the amino acid sequence "TEKCPDPYKPKPKPTPKPTPTPYPSAGKCPRDALKLGVCADVLNLVHNVVIGSPPTLPCCSLLEGLVNLEAAVCLCTAIKANILGKNLNLPIALSLVLNNCGKQVPNGFECT" . The 46 kDa protein likely contains similar motifs but with additional structural elements accounting for its larger molecular weight.

What analytical methods are recommended for initial confirmation of recombinant Daucus carota 46 kDa cell wall protein expression?

For initial confirmation of recombinant expression, a combination of PCR and Western blot analysis is recommended. PCR with gene-specific primers confirms successful integration of the gene into expression vectors or host genomes, as demonstrated in transgenic carrot studies where PCR confirmed the presence of integrated sequences . Western blot analysis using polyclonal antibodies raised against the protein or related epitopes provides confirmation of protein expression. For instance, studies have successfully used antisera raised against synthetic peptides predicted to have homology with unhydroxylated, unglycosylated precursors of cell wall proteins to detect carrot cell wall proteins in immunological studies .

What are the optimal expression systems for producing recombinant Daucus carota 46 kDa cell wall protein?

Based on research with similar carrot proteins, E. coli represents a viable heterologous expression system for recombinant carrot cell wall proteins, particularly when expressing non-glycosylated forms . When proper glycosylation is required for functional studies, plant-based expression systems are preferable. Transgenic carrot systems have been successfully used to express recombinant proteins, as evidenced by studies where foreign genes were integrated into the carrot nuclear genome . For the 46 kDa protein specifically, the choice between prokaryotic (E. coli) and eukaryotic (plant-based) expression systems depends on whether post-translational modifications are necessary for the research objectives.

What purification strategies are most effective for isolating recombinant Daucus carota 46 kDa cell wall protein while maintaining its native structure?

Effective purification of recombinant carrot cell wall proteins typically involves affinity chromatography, particularly when the recombinant protein includes an affinity tag like the His-tag used for the 14 kDa proline-rich protein . For maintaining native structure, consider these parameters:

• Buffer composition: Tris/PBS-based buffer with pH 8.0 has been successfully used for similar carrot proteins

• Stabilizers: Addition of 6% trehalose helps maintain protein stability during storage

• Reconstitution: Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Storage: Add 5-50% glycerol (final concentration) and store at -20°C/-80°C in small aliquots to avoid repeated freeze-thaw cycles

How can researchers optimize the yield of soluble recombinant Daucus carota 46 kDa cell wall protein in E. coli expression systems?

To optimize soluble protein yield in E. coli, researchers should consider implementing stress-tolerant expression strategies. Research with carrot heat shock proteins has demonstrated that expression of plant stress proteins like Hsp17.7 in E. coli can increase soluble protein levels by up to 20% under stress conditions . Specific optimization strategies include:

• Temperature modulation: Lowering expression temperature to 16-20°C to reduce inclusion body formation

• Co-expression with chaperones: Introducing molecular chaperones to assist protein folding

• Fusion tags: Utilizing solubility-enhancing fusion partners such as MBP (maltose-binding protein)

• Promoter selection: The lipoprotein (Lpp) gene promoter has shown efficacy for expressing plant proteins in E. coli

• Induction optimization: Adjusting IPTG concentration and induction time based on experimental validation

How do post-translational modifications affect the structure and function of the Daucus carota 46 kDa cell wall protein?

Post-translational modifications, particularly proline hydroxylation and glycosylation, significantly impact the structure and function of carrot cell wall proteins. Research using the proline hydroxylase inhibitor α,α'-dipyridyl has shown that inhibiting hydroxylation prevents normal glycosylation of carrot cell wall proteins, resulting in a non-hydroxylated, non-glycosylated form that still targets to the cell wall . The native 46 kDa protein likely undergoes hydroxylation of proline residues followed by O-linked arabinosylation, which affects protein conformation, solubility, and resistance to proteolytic degradation. Interestingly, studies have shown that even without hydroxylation and arabinosylation, carrot cell wall proteins can still become covalently attached to the cell wall matrix, suggesting that these modifications are not absolute requirements for cell wall integration .

What methods can be used to analyze glycosylation patterns of recombinant Daucus carota 46 kDa cell wall protein?

To analyze glycosylation patterns of the recombinant protein, researchers should employ a combination of analytical techniques:

• Radioactive labeling: Incorporation of radioactive precursors (e.g., [³H]proline or [³H]arabinose) followed by analysis of secreted proteins, as demonstrated in carrot disc systems

• Lectin affinity analysis: Using plant lectins with specificity for different glycan structures

• Mass spectrometry: NanoLC-ESI-MS/MS for detailed identification of glycopeptides and glycan structures, as used for identifying carrot root proteins

• Comparative mobility analysis: SDS-PAGE comparison of glycosylated versus deglycosylated forms following enzymatic deglycosylation

• Inhibitor studies: Using glycosylation inhibitors to produce partially glycosylated forms for structural/functional comparison

How can researchers distinguish between native and recombinant forms of the Daucus carota 46 kDa cell wall protein?

Distinguishing between native and recombinant forms requires multiple analytical approaches:

• Western blot analysis using antibodies that recognize epitopes present in both forms, similar to approaches used with synthetic peptide antisera for identifying carrot cell wall proteins

• Mass spectrometry to detect differences in post-translational modifications or the presence of expression tags

• Comparison of electrophoretic mobility in SDS-PAGE, as glycosylation affects apparent molecular weight

• Peptide mapping following protease digestion to identify sequence differences or modifications

• Analysis of amino acid composition, particularly hydroxylated proline content, which differs between plant-produced and bacterially-expressed proteins

What experimental approaches are recommended for studying the interaction of recombinant Daucus carota 46 kDa cell wall protein with other cell wall components?

For studying protein-cell wall component interactions, consider these methodological approaches:

• In vitro binding assays with isolated cell wall polymers (cellulose, hemicellulose, pectin)

• Carrot disc systems with radioactive labeling to track protein integration into cell walls under various conditions

• Immunolocalization using antibodies against the 46 kDa protein to visualize its distribution within cell wall structures

• Cross-linking studies to identify protein-protein or protein-polysaccharide interactions

• Competitive binding assays with known cell wall proteins to identify shared binding sites

These approaches can reveal whether the recombinant protein maintains native binding capacity and properly integrates into cell wall architecture.

How does the recombinant Daucus carota 46 kDa cell wall protein respond to environmental stresses compared to its native counterpart?

Understanding stress responses requires comparative analysis between recombinant and native proteins:

• Exposure to abiotic stresses: Studies with carrot root cells under boron excess stress have identified several defense-related proteins, including CR16 (Major allergen Dau c1) and glutathione peroxidase . Similar approaches can be applied to study the 46 kDa protein's response.

• Differential expression analysis: Comparing protein levels in stressed versus non-stressed conditions

• Functional complementation: Testing whether the recombinant protein can restore stress tolerance in knockout/knockdown plant lines

• Post-translational modification analysis: Determining if stress conditions alter modification patterns

• Stability assays: Comparing thermal stability and resistance to proteolytic degradation between native and recombinant forms under stress conditions

What role does the Daucus carota 46 kDa cell wall protein play in plant defense mechanisms?

The potential role in plant defense can be investigated through:

• Challenge experiments: Exposing transgenic plants overexpressing the 46 kDa protein to pathogens

• Protein interaction studies: Identifying binding partners related to defense signaling

• Comparative analysis with known defense proteins: Several carrot proteins involved in defense mechanisms have been identified (glutathione peroxidase, glyoxylase I, isocitrate dehydrogenase) that could serve as comparators

• Recombinant protein application: Testing if external application of the recombinant protein induces defense responses

• Expression profiling: Analyzing the timing and localization of protein expression during pathogen attack

How can the recombinant Daucus carota 46 kDa cell wall protein be used as a model for studying cell wall protein evolution across plant species?

For evolutionary studies, researchers should consider:

• Comparative sequence analysis: Aligning the 46 kDa protein sequence with homologs from other plant species

• Structural modeling: Predicting three-dimensional structures and comparing conserved domains

• Functional conservation testing: Expressing the recombinant protein in heterologous plant systems to test for functional complementation

• Phylogenetic analysis: Constructing evolutionary trees based on sequence similarity and structural features

• Domain-swapping experiments: Creating chimeric proteins with domains from different species to identify functionally critical regions

Such studies can reveal evolutionary relationships between carrot cell wall proteins and homologs in other plant species.

What are the latest methodological advances for studying the structure-function relationship of the Daucus carota 46 kDa cell wall protein?

Current advanced methodologies include:

• Cryo-electron microscopy for high-resolution structural analysis

• CRISPR-Cas9 gene editing for creating precise mutations in the native gene

• Single-molecule force spectroscopy to measure protein-cell wall binding dynamics

• Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions and binding interfaces

• Computational molecular dynamics simulations to predict structural changes under different conditions

• In situ hybridization combined with immunolocalization to correlate gene expression with protein localization

These techniques allow for detailed structure-function analyses beyond traditional biochemical approaches.

How can researchers design mutational studies to identify critical functional domains in the Daucus carota 46 kDa cell wall protein?

For effective mutational analysis:

• Design a series of deletion mutants targeting predicted functional domains

• Create site-directed mutations at conserved amino acid residues

• Focus on proline-rich regions, as these are likely crucial for structure and function based on studies of similar proteins like the 14 kDa proline-rich protein

• Target potential hydroxylation and glycosylation sites identified through bioinformatic analysis

• Construct chimeric proteins by swapping domains with other cell wall proteins

• Express mutant proteins in both prokaryotic (E. coli) and eukaryotic systems to assess the impact of post-translational modifications on mutant phenotypes

What are common challenges in expressing recombinant Daucus carota 46 kDa cell wall protein and how can they be addressed?

Common challenges and solutions include:

• Insoluble protein expression: Optimize by lowering expression temperature or using solubility tags; alternatively, integrate stress-tolerant systems like those using Hsp17.7

• Improper folding: Co-express with molecular chaperones or use periplasmic expression systems

• Degradation during purification: Include protease inhibitors and optimize buffer conditions similar to those used for the 14 kDa proline-rich protein (Tris/PBS-based buffer with 6% trehalose at pH 8.0)

• Low expression levels: Optimize codon usage for the expression host or try alternative promoters such as the lipoprotein (Lpp) gene promoter used successfully with carrot proteins

• Lack of post-translational modifications: If required, switch from prokaryotic to eukaryotic expression systems such as transgenic carrot plants

How should researchers address contradictory results when comparing native versus recombinant Daucus carota 46 kDa cell wall protein functions?

When facing contradictory results:

• Verify protein identity and integrity through mass spectrometry and sequencing

• Systematically compare post-translational modifications between native and recombinant forms

• Assess the impact of affinity tags on protein function through tag removal experiments

• Examine expression system artifacts that might affect protein folding or activity

• Consider using multiple expression systems to confirm that observed differences are consistent

• Validate antibody specificity when using immunological detection methods, as demonstrated in carrot cell wall protein studies

What quality control measures should be implemented when working with recombinant Daucus carota 46 kDa cell wall protein preparations?

Essential quality control measures include:

• Purity assessment: Confirm >90% purity by SDS-PAGE, similar to standards used for other recombinant carrot proteins

• Identity confirmation: Verify protein identity by Western blot and mass spectrometry, as used in carrot protein identification studies

• Stability monitoring: Implement periodic testing of stored protein samples to detect degradation

• Functional assays: Develop reproducible activity assays specific to known or predicted functions

• Batch consistency: Compare multiple expression batches to ensure reproducibility

• Endotoxin testing: For proteins intended for immunological studies, confirm removal of bacterial endotoxins

• Storage validation: Verify protein stability under recommended storage conditions (-20°C/-80°C with 50% glycerol)