Recombinant Solanum lycopersicum 35 kDa cell wall protein

What are the major cell wall proteins found in Solanum lycopersicum in the 35 kDa range?

Several important cell wall proteins in tomato (Solanum lycopersicum) have molecular weights around 35 kDa, including pectin methylesterases (PMEs), receptor-like kinases, and various glycosyltransferases. Proteomic profiling has identified these proteins in cell wall-enriched fractions from various plant tissues. For example, pectin methylesterases are commonly observed at approximately 35 kDa in SDS-PAGE analysis, which is consistent with the predicted size of fully processed (mature) PME proteins . These proteins play crucial roles in cell wall assembly, remodeling, and signaling pathways critical for plant development and defense responses.

How can I confirm the identity of a putative 35 kDa tomato cell wall protein?

Confirmation of a putative 35 kDa tomato cell wall protein requires multiple complementary approaches:

• Mass spectrometry analysis: Perform tryptic digestion of the purified protein band from SDS-PAGE and analyze peptide fragments. Match the detected peptides against databases of known tomato proteins. For reliable identification, aim for at least 10-12 matching peptides, similar to the approach used for AtPME2 identification which matched 12 peptides .

• Western blot analysis: Use antibodies specific to conserved domains found in the suspected protein family. For example, antibodies generated against the highly conserved region V in plant PMEs (e.g., sequence KTYLGRPWKEYSRTV) can detect multiple PME isoforms .

• Functional assays: Perform enzymatic activity tests specific to the suspected protein function to confirm its identity beyond sequence homology.

• Northern blot analysis: Verify RNA production of the appropriate molecular weight in the tissue of interest, especially when working with recombinant proteins .

What expression systems are most effective for producing recombinant Solanum lycopersicum cell wall proteins?

The effectiveness of expression systems for tomato cell wall proteins varies based on the specific protein and research objectives:

Plant-based expression systems:

• Transgenic tomato plants: Provide native post-translational modifications and processing. Success has been demonstrated with foreign proteins like rabies virus glycoprotein expressed in tomato plants under the control of the 35S promoter of cauliflower mosaic virus .

• Arabidopsis thaliana: Useful as a model system for studying wall proteins, particularly when exploring functional homology .

Non-plant expression systems:

• Pichia pastoris: Effectively produces plant cell wall proteins, including PMEs, with proper folding and post-translational modifications. This system successfully produced mature active AtPME2 enzyme (~35 kDa) along with the PRO-peptide (~30 kDa) .

• E. coli with specialized vectors: Using thioredoxin fusion proteins can improve solubility of plant cell wall proteins that are otherwise difficult to express .

Each system has advantages and limitations, with the optimal choice depending on the protein characteristics and experimental requirements.

What purification strategy should I use to isolate recombinant Solanum lycopersicum cell wall proteins?

A systematic purification strategy integrating multiple techniques yields the best results:

• Initial extraction: For cell wall proteins, use buffers containing salt (0.2-1.0 M NaCl) to release ionically bound proteins from the cell wall matrix.

• Chromatography selection based on protein properties:

  • Cation exchange chromatography: Particularly effective for PMEs and other basic cell wall proteins, as demonstrated in the purification of recombinant AtPME2 .

  • Affinity chromatography: Consider using epitope tags if the native protein function allows.

  • Size exclusion chromatography: Useful as a polishing step to achieve high purity.

• Verification of purity:

  • SDS-PAGE with Coomassie-Blue staining to visualize protein bands

  • Western blot using specific antibodies to confirm identity

  • Mass spectrometry to verify sequence and post-translational modifications

When examining purified fractions, be aware that plant cell wall proteins often appear as multiple bands due to differential processing. For example, AtPME2 shows bands at both ~30 kDa and ~35 kDa due to cleavage at different processing motifs (RKLK and RRLL) .

How do post-translational modifications affect the function of recombinant tomato cell wall proteins?

Post-translational modifications (PTMs) critically influence the function of tomato cell wall proteins through several mechanisms:

• Proteolytic processing: Many cell wall proteins, including PMEs, are synthesized as precursors and require proteolytic cleavage for activation. The presence of processing motifs (such as RKLK and RRLL in AtPME2) results in mature proteins of approximately 35 kDa . Incorrect processing in heterologous systems can lead to inactive proteins or altered activity.

• Phosphorylation: Regulates protein activity and interactions, particularly for receptor-like kinases. For example, SlFERL in tomato triggers downstream signaling by phosphorylating SlMAP3K18 at specific residues (Thr45, Ser49, Ser76, and Ser135), which is essential for immune responses to pathogens .

• Glycosylation: Affects protein stability, folding, and cell wall targeting. Expression in systems lacking proper glycosylation machinery can result in misfolded or mistargeted proteins.

When designing experiments with recombinant tomato cell wall proteins, researchers should carefully consider the expression system's ability to perform the necessary PTMs for proper protein function.

What methods are recommended for assessing the activity of recombinant 35 kDa tomato cell wall proteins?

Activity assessment methods should be tailored to the specific protein class:

For pectin methylesterases (PMEs):

• pH-dependent activity assays: Monitor de-esterification of pectin at different pH values to determine pH optima and processivity patterns .

• Gel diffusion assays: Use ruthenium red staining to visualize zones of de-methylesterified pectin.

• Degree of methyl-esterification (DM) measurement: Quantify methanol release using colorimetric or HPLC-based methods.

For receptor-like kinases:

• Phosphorylation assays: Using radioactive ATP (γ-32P-ATP) or phospho-specific antibodies to detect kinase activity.

• Protein-protein interaction studies: Co-immunoprecipitation to identify binding partners, as demonstrated with SlFERL and BcPG1 .

• Functional complementation: Test the ability of the recombinant protein to rescue mutant phenotypes.

For glycosyltransferases:

• Radiochemical assays: Measure incorporation of radiolabeled sugar donors into acceptor molecules.

• HPLC or mass spectrometry: Analyze reaction products to verify glycosyltransferase activity.

How can I design experiments to study the role of 35 kDa cell wall proteins in tomato immune responses?

Designing experiments to investigate the role of 35 kDa cell wall proteins in tomato immune responses requires a multi-faceted approach:

• Genetic manipulation strategies:

  • CRISPR/Cas9 gene editing: Create precise knockouts or modifications of target genes.

  • RNAi or antisense suppression: For partial knockdown when complete knockout is lethal.

  • Overexpression studies: Express the protein under constitutive promoters to observe gain-of-function phenotypes.

• Pathogen challenge experiments:

  • Test responses to relevant pathogens like Botrytis cinerea, which is known to interact with tomato cell wall proteins .

  • Quantify disease progression through lesion size measurements, fungal biomass determination, and expression analysis of defense genes.

  • Compare responses in wild-type versus genetically modified plants under controlled conditions.

• Signaling pathway analysis:

  • Monitor changes in MAPK signaling cascades, as seen with SlFERL-mediated immune responses .

  • Measure ROS production using luminol-based assays or H₂O₂-specific probes.

  • Analyze hormone production (salicylic acid, jasmonic acid, ethylene) in response to elicitation.

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation experiments to identify interacting partners.

  • Use yeast two-hybrid or bimolecular fluorescence complementation to confirm direct interactions.

  • Consider structural biology approaches (X-ray crystallography, cryo-EM) for detailed interaction mechanisms.

The SlFERL study provides a valuable framework, showing how a receptor-like kinase recognizes pathogen proteins (BcPG1) and modulates downstream MAPK signaling pathways .

What challenges exist in expressing and purifying recombinant tomato cell wall proteins, and how can they be overcome?

Researchers face several significant challenges when working with recombinant tomato cell wall proteins:

A systematic pipeline approach as described in search result can help overcome these challenges through:

• Fractional experimental design to efficiently screen multiple variables

• Testing various expression vectors (intracellular, periplasmic, fusion proteins)

• Optimizing growth conditions (temperature, induction timing, media composition)

• Employing a high-throughput screening system to identify successful conditions

Researchers have successfully applied these strategies to express and purify plant cell wall glycosyltransferases in unprecedented milligram amounts , suggesting similar approaches could work for tomato cell wall proteins.

How do tomato 35 kDa cell wall proteins compare to their homologs in Arabidopsis and other model plants?

Tomato (Solanum lycopersicum) 35 kDa cell wall proteins share both conservation and divergence with their homologs in Arabidopsis and other plant species:

• Structural conservation:

  • Catalytic domains of enzymes like PMEs show high sequence conservation across plant species, particularly in functional regions. For example, region V in plant PMEs contains the highly conserved sequence KTYLGRPWKEYSRTV that includes catalytic residues .

  • Receptor-like kinases such as SlFERL in tomato share structural organization with Arabidopsis homologs, containing extracellular, transmembrane, and kinase domains .

• Functional specialization:

  • Despite sequence similarity, tomato cell wall proteins often show species-specific functions related to fruit development and softening, which are less prominent in non-fleshy fruit plants like Arabidopsis.

  • Proteomic comparisons between species reveal that while some proteins are common across plants (e.g., AT3G14310, AT4G19410), others appear to be species-specific or have differential expression patterns .

• Evolutionary adaptations:

  • Tomato receptor-like kinases involved in pathogen recognition, such as SlFERL, have evolved specificity for recognizing pathogens that commonly infect Solanaceae crops .

  • Gene duplication and diversification have resulted in expanded families of cell wall proteins, with specialized functions compared to their Arabidopsis counterparts.

When conducting comparative studies, researchers should consider both the conserved domains for functional predictions and the species-specific adaptations that may impact experimental outcomes.

What insights can be gained from studying recombinant versus native tomato cell wall proteins?

Comparative analysis of recombinant versus native tomato cell wall proteins provides valuable insights into protein function, regulation, and biotechnological applications:

• Post-translational modifications:

  • Native proteins often contain complex patterns of glycosylation and other modifications that may be absent or different in recombinant systems.

  • Comparing activity of native versus recombinant proteins can reveal the functional importance of these modifications.

• Protein complexes and interactions:

  • Native proteins may exist in multi-protein complexes that are difficult to reconstitute in recombinant systems.

  • For example, cellulose synthase complexes in plant cell walls involve multiple CESA proteins arranged in specific stoichiometry (Class I proteins CESA1, CESA3, and any one of CESA 2, 5, 6, or 9 for primary cell wall synthesis) .

• Enzymatic behavior:

  • pH-dependent activity profiles may differ between recombinant and native proteins due to differences in protein folding or interaction partners.

  • The processivity patterns of enzymes like PMEs can vary depending on expression system and purification method .

• Experimental advantages of recombinant systems:

  • Site-directed mutagenesis of recombinant proteins allows precise mapping of functional residues.

  • For example, mutation studies of SlFERL phosphorylation sites (Thr45, Ser49, Ser76, and Ser135) revealed their crucial role in immune signaling pathways .

• Biotechnological applications:

  • Recombinant expression enables production of modified tomato cell wall proteins with enhanced stability or novel activities.

  • Examples include engineering tomato plants to express foreign proteins like the rabies virus glycoprotein, showing the potential for plants to serve as expression systems .

What emerging technologies are advancing research on recombinant tomato cell wall proteins?

Several cutting-edge technologies are transforming research on recombinant tomato cell wall proteins:

• CRISPR/Cas9 genome editing:

  • Allows precise modification of native tomato genes encoding cell wall proteins.

  • Enables creation of protein variants with altered domains, processing sites, or regulatory elements.

  • Facilitates rapid generation of knockout lines for functional studies.

• Cryo-electron microscopy:

  • Provides structural insights into cell wall protein complexes at near-atomic resolution.

  • Recent advances in sample preparation and detector technology make it possible to visualize membrane-associated cell wall protein assemblies.

  • Particularly valuable for understanding protein complexes like cellulose synthase that are difficult to crystallize .

• Single-molecule techniques:

  • Enable real-time observation of enzyme processivity and mechanisms.

  • Fluorescence resonance energy transfer (FRET) approaches allow monitoring of protein-protein interactions in living cells.

• Advanced mass spectrometry:

  • Top-down proteomics provides comprehensive analysis of intact proteins, revealing the full spectrum of post-translational modifications.

  • Crosslinking mass spectrometry identifies interaction surfaces between cell wall proteins and their partners.

• Cell-free expression systems:

  • Allow rapid screening of protein variants without time-consuming transformation and cultivation steps.

  • Can be coupled with microfluidic platforms for high-throughput optimization of expression conditions .

These technologies collectively provide unprecedented opportunities to understand the structure, function, and regulation of tomato cell wall proteins.

How can high-throughput approaches be optimized for studying tomato cell wall protein expression and function?

Optimizing high-throughput approaches for tomato cell wall protein research requires strategic integration of several methodologies:

• Expression screening optimization:

  • Implement fractional experimental design to efficiently explore the multidimensional parameter space affecting protein expression .

  • Key parameters to screen include:

    • Vector design (signal peptides, fusion partners, solubility tags)

    • Expression hosts (bacterial, yeast, insect cell, plant-based)

    • Induction conditions (temperature, inducer concentration, time)

    • Co-expression partners (chaperones, modifying enzymes)

• Functional characterization pipeline:

  • Develop multiplexed activity assays compatible with 96 or 384-well formats.

  • Establish automated image analysis for phenotypic assessment of mutant populations.

  • Utilize robot-assisted sample preparation to reduce variability and increase throughput.

• Data integration strategies:

  • Implement machine learning approaches to identify patterns in expression and activity data.

  • Develop predictive models to guide expression optimization for new protein targets.

  • Create standardized data formats to facilitate comparison across different laboratories.

• Validation approaches:

  • Design confirmatory experiments to verify hits from high-throughput screens.

  • Establish quantitative metrics for success (protein yield, purity, specific activity).

  • Use reference proteins with known properties as internal controls.

The pipeline approach described in search result provides a valuable framework, demonstrating how high-throughput expression screening can help overcome the challenges inherent in producing difficult-to-express plant cell wall proteins.