Recombinant Solanum lycopersicum 23 kDa cell wall protein

What is the 23 kDa cell wall protein from Solanum lycopersicum?

The 23 kDa cell wall protein from Solanum lycopersicum is a secreted protein localized to the plant cell wall. According to UniProtKB/Swiss-Prot classification, it is identified as CWP25_SOLLC (P80821). The protein was characterized through differential extraction and protein sequencing methods that revealed distinct patterns of primary cell wall proteins from plants. Currently, only a fragment of 20 amino acids has been fully sequenced according to database entries . The protein was initially studied by Robertson et al. in their 1997 research focused on differential extraction methodologies for plant cell wall proteins .

How does this protein compare structurally to other plant cell wall proteins?

While complete structural information for the 23 kDa tomato cell wall protein remains limited, it shares characteristics with other secreted cell wall proteins. As a secreted protein targeted to the cell wall , it likely contains signal peptides that direct its localization. Similar to other plant cell wall proteins, it may have domains involved in interactions with polysaccharides or other cell wall components. The molecular weight (23 kDa) places it in the category of smaller cell wall proteins, distinguishing it from larger structural proteins like extensins or glycine-rich proteins frequently found in plant cell walls.

What methodologies are most effective for initial characterization of this protein?

For comprehensive initial characterization of the 23 kDa cell wall protein, researchers should implement a multi-method approach:

• Extraction optimization: Using differential extraction methods as described by Robertson et al. to isolate the native protein from tomato cell walls .

• Protein sequencing: Complete protein sequencing through a combination of Edman degradation and mass spectrometry approaches to resolve the full amino acid sequence beyond the currently known 20-amino acid fragment .

• Recombinant expression screening: Implementation of high-throughput expression screening approaches similar to those developed for plant cell wall glycosyltransferases, testing multiple expression systems, fusion partners, and conditions .

• Subcellular localization: Immunolocalization or fluorescent protein fusion approaches to confirm and precisely map cell wall localization within different plant tissues.

What expression systems are most suitable for recombinant production of this protein?

Based on research with similar plant proteins, several expression systems show promise for recombinant production of the 23 kDa tomato cell wall protein:

• Plant-based expression systems: Transient expression in Solanum lycopersicum via agroinfiltration has demonstrated success for recombinant protein production in tomato, allowing native post-translational modifications and proper folding . This homologous expression approach may be particularly advantageous for cell wall proteins.

• E. coli expression systems: Bacterial expression can be effective but typically results in low soluble:insoluble ratios for plant cell wall proteins. Success can be improved through co-expression with chaperones (DnaK/DnaJ/GrpE and Trigger Factor) and optimization of lysis buffer composition .

• Eukaryotic alternatives: For challenging cases, yeast or insect cell expression systems might provide appropriate cellular machinery for proper folding and post-translational modifications.

The choice should be guided by research requirements, particularly regarding native modifications and functional studies.

What strategies can improve solubility of the recombinant 23 kDa cell wall protein?

Optimizing solubility for recombinant plant cell wall proteins represents a significant challenge. Research with plant cell wall glycosyltransferases provides valuable insights applicable to the 23 kDa protein:

As noted in studies with plant cell wall glycosyltransferases, "recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures" , necessitating systematic optimization of these factors.

How can high-throughput screening approaches be applied to optimize expression?

Implementing a high-throughput screening pipeline similar to that developed for plant cell wall glycosyltransferases can efficiently identify optimal expression conditions:

• Construct design: Create a library of expression constructs with variations in:

  • N-terminal truncations to remove potential transmembrane domains

  • Different fusion partners (His-tag, MBP, GST, TRX)

  • Periplasmic vs. cytoplasmic targeting sequences

• Expression condition matrix: Systematically test combinations of:

  • E. coli strains (BL21, Rosetta, Origami, SHuffle)

  • Induction temperatures (15°C, 25°C, 37°C)

  • Inducer concentrations (0.1-1.0 mM IPTG)

  • Co-expression with various chaperone systems

• Fractional experimental design: Implement a strategic experimental design to test multiple variables without exhaustive combinations, as described for glycosyltransferase screening .

• Automated analysis: Develop high-throughput solubility assays to rapidly assess soluble:insoluble ratios across conditions.

This systematic approach can identify conditions yielding the highest levels of soluble, correctly folded protein for subsequent scale-up and characterization.

What purification strategy is most effective for the recombinant 23 kDa cell wall protein?

A multi-step purification approach is recommended for obtaining high-purity recombinant 23 kDa tomato cell wall protein:

• Initial capture: Affinity chromatography using fusion tags (His-tag, GST) provides efficient initial purification.

• Intermediate purification: Ion-exchange chromatography to separate the target protein based on charge properties.

• Polishing step: Reverse-phase HPLC using C4 columns has proven effective for achieving final purity of similar proteins, as demonstrated in the purification of other protein targets .

• Quality control: Size-exclusion chromatography to verify homogeneity and remove aggregates.

For each step, optimization of buffer conditions (pH, ionic strength, additives) is critical for maintaining protein stability and maximizing recovery.

How can researchers confirm proper folding and post-translational modifications?

Verification of proper folding and post-translational modifications requires multiple complementary approaches:

• Structural analysis:

  • Circular dichroism spectroscopy to assess secondary structure

  • Limited proteolysis patterns compared to native protein

  • Thermal stability assays (differential scanning fluorimetry)

• Post-translational modification analysis:

  • Mass spectrometry for comprehensive PTM mapping

  • Glycan analysis if the protein is glycosylated

  • Site-directed mutagenesis of potential modification sites

• Functional verification:

  • Binding assays with potential cell wall interaction partners

  • Complementation of mutant phenotypes in planta

  • Immunological recognition by conformation-specific antibodies

These approaches should be selected based on available resources and specific research questions regarding the protein's structure-function relationships.

What analytical methods are most informative for studying protein-cell wall interactions?

Understanding how the 23 kDa protein interacts with other cell wall components requires specialized analytical approaches:

• In vitro binding assays:

  • Surface plasmon resonance (SPR) to measure binding kinetics with purified cell wall components

  • Isothermal titration calorimetry (ITC) for thermodynamic analysis of interactions

  • Pull-down assays using immobilized cell wall polysaccharides

• Microscopy techniques:

  • Immunogold electron microscopy for precise localization within cell wall ultrastructure

  • Förster resonance energy transfer (FRET) to detect protein-protein interactions in situ

  • Super-resolution microscopy for detailed spatial distribution analysis

• Biochemical approaches:

  • Cross-linking studies followed by mass spectrometry to identify interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Sequential extraction analysis to determine strength of cell wall associations

These methods provide complementary information about the protein's structural integration and functional interactions within the complex cell wall matrix.

How can researchers determine the biological function of the 23 kDa cell wall protein?

Elucidating the biological function requires a systematic research strategy combining genetic, biochemical, and physiological approaches:

• Genetic manipulation:

  • CRISPR/Cas9 gene editing to generate knockout mutants

  • RNAi or antisense strategies for knockdown studies

  • Overexpression lines to assess gain-of-function phenotypes

• Phenotypic analyses:

  • Cell wall composition analysis (monosaccharide composition, linkage analysis)

  • Mechanical property testing (extensibility, strength)

  • Growth and developmental phenotyping under various conditions

• Comparative proteomics:

  • Identify changes in the cell wall proteome in mutant/transgenic lines

  • Compare expression patterns across developmental stages and stress responses

• Complementation studies:

  • Rescue mutant phenotypes with wild-type gene to confirm specificity

  • Domain swap experiments to identify functional regions

This multi-faceted approach can provide convergent evidence for the protein's biological role in cell wall architecture, development, or stress responses.

What role might this protein play in plant development and stress responses?

While specific functional data for the 23 kDa tomato cell wall protein is limited in the available literature, its localization to the cell wall suggests several potential roles:

• Cell expansion and development:

  • Regulation of cell wall extensibility during growth

  • Involvement in fruit development and ripening processes specific to tomato

• Cell wall integrity:

  • Structural reinforcement of cell walls

  • Participation in cell wall remodeling during development

• Stress responses:

  • Potential involvement in pathogen resistance through cell wall reinforcement

  • Possible role in abiotic stress responses requiring cell wall modification

Research investigating expression patterns during development, fruit ripening, and under various stress conditions would provide valuable insights into the protein's physiological significance.

How does post-translational processing affect the function of this protein?

Post-translational processing likely plays a critical role in the function of the 23 kDa cell wall protein, though specific modifications remain to be characterized:

• Signal peptide processing: As a secreted protein, removal of the N-terminal signal peptide is essential for proper localization to the cell wall .

• Potential glycosylation: Many cell wall proteins undergo glycosylation, which can affect:

  • Protein stability and protection from proteolysis

  • Interaction with cell wall polysaccharides

  • Recognition by other proteins in signaling pathways

• Hydroxylation and other modifications: Cell wall proteins often undergo proline hydroxylation or other modifications that influence their structural properties.

• Cross-linking potential: Some cell wall proteins participate in cross-linking reactions that integrate them into the wall matrix.

Characterization of these modifications and their functional significance represents an important research area that could be addressed through comparative analysis of native and recombinant proteins using mass spectrometry and functional assays.

What are the major challenges in expressing and purifying this recombinant protein?

Research with plant cell wall proteins reveals several significant challenges likely applicable to the 23 kDa tomato cell wall protein:

• Low solubility: "Recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures" , a limitation potentially shared by structural cell wall proteins.

• Proper folding: "Correct folding of CWGTs in heterologous expression hosts is problematic and this bottleneck can be partly alleviated by co-expression with chaperones" . Similar folding challenges may affect the 23 kDa protein.

• Post-translational modifications: Achieving native modifications in heterologous systems can be difficult, potentially affecting protein function and stability.

• Proteolytic degradation: Cell wall proteins may be susceptible to proteolysis during extraction and purification.

• Yield limitations: Even with optimization, yields of correctly folded, soluble protein may remain relatively low compared to cytosolic proteins.

Addressing these challenges requires systematic optimization of expression conditions, buffer compositions, and purification strategies.

How can researchers overcome protein aggregation during recombinant expression?

Protein aggregation represents a common challenge in recombinant expression of plant cell wall proteins. Strategies to minimize aggregation include:

• Expression optimization:

  • Reduce expression temperature to 15-20°C during induction

  • Decrease inducer concentration to slow protein synthesis

  • Consider auto-induction media for gradual protein production

• Solubilizing agents:

  • Include low concentrations of mild detergents in lysis buffers

  • Test additives like arginine, proline, or sucrose as chemical chaperones

  • Optimize salt concentration to maintain protein solubility

• Co-expression strategies:

  • Co-express with molecular chaperones such as DnaK/DnaJ/GrpE and Trigger Factor, which "seemed to enhance soluble CWGT production"

  • Consider co-expression with specific binding partners if known

• Protein engineering:

  • Identify and modify aggregation-prone regions through predictive algorithms

  • Create fusion constructs with highly soluble partners

  • Consider surface charge modifications to enhance solubility

Systematic testing of these approaches, potentially using a fractional experimental design as described for plant cell wall glycosyltransferases , can identify optimal conditions for minimizing aggregation.

What control experiments are essential when working with this recombinant protein?

Rigorous control experiments are crucial for reliable research with the recombinant 23 kDa cell wall protein:

• Expression validation controls:

  • Empty vector controls processed identically to expression cultures

  • Western blotting with tag-specific and protein-specific antibodies

  • Mass spectrometry verification of protein identity

• Functional assay controls:

  • Heat-denatured protein controls to verify activity requires native conformation

  • Enzymatically treated protein (e.g., deglycosylation) to assess modification importance

  • Concentration-dependent activity measurements to establish specificity

• Localization controls:

  • Free fluorescent protein controls for fusion protein localization studies

  • Competitive binding controls for antibody-based detection

  • Fractionation controls with known markers for different cellular compartments

• Interaction study controls:

  • Negative control proteins with similar properties but different functions

  • Competition assays with unlabeled protein to verify binding specificity

  • Mutated binding site variants to confirm interaction mechanisms