Recombinant Solanum lycopersicum 44 kDa cell wall protein 2

What is the biological function of the 44 kDa cell wall protein in Solanum lycopersicum?

The 44 kDa protein in tomato appears to be a chitinase (PR-3) that functions as part of the plant's defense mechanism. Western blot analysis has revealed the presence of this 44 kDa chitinase in elicitor-treated suspension-cultured cells and leaves of tomato . As a pathogenesis-related protein, it plays a crucial role in the plant's immune response by degrading chitin, a major component of fungal cell walls. The expression of this protein is significantly upregulated during pathogen attack or when treated with elicitors, indicating its importance in disease resistance. Cell wall proteins like chitinase are essential for maintaining cell wall integrity while simultaneously participating in defense against invading pathogens .

How is the expression of this cell wall protein regulated during tomato development?

The expression of cell wall proteins in tomatoes, including the 44 kDa chitinase, is regulated by complex transcriptional networks. The MADS domain transcription factor RIN (ripening inhibitor) plays a critical role by directly binding to the promoters of several cell wall-related genes, including LeEXP1, which is involved in cell wall development . Proteomic analysis has revealed that in ripening tomatoes, proteins involved in cell wall development (including LeEXP1), ethylene biosynthesis, and ABA signaling pathways show significant changes in abundance . Additionally, virus-induced gene silencing experiments have demonstrated that silencing specific transcription factors can inhibit the expression of defense-related proteins, suggesting a coordinated regulation of these proteins during both development and stress responses .

What structural features characterize the 44 kDa cell wall protein?

While specific structural details of the tomato 44 kDa chitinase are not fully described in the provided literature, this protein likely shares common structural features with other plant chitinases. As a member of the PR-3 family, it would contain a catalytic domain responsible for hydrolyzing the β-1,4-glycosidic bonds in chitin polymers. The protein likely possesses multiple disulfide bonds that contribute to its stability and proper folding, similar to other defense proteins in plants . Western blot analysis has confirmed that this protein can be detected alongside a 23 kDa thaumatin-like protein (PR-5) in tomato tissues following elicitor treatment, suggesting they may work in concert as part of the plant's defense arsenal .

How does the 44 kDa chitinase compare with other antimicrobial proteins found in tomatoes?

Tomatoes produce several antimicrobial proteins as part of their defense system. The 44 kDa chitinase (PR-3) works alongside other pathogenesis-related proteins including thaumatin-like proteins (PR-5) and β-1,3-glucanases, which are induced upon pathogen challenge or elicitor treatment . More recently characterized is SlHBP2, a SOUL heme-binding family protein that exhibits significant antimicrobial activity against bacterial pathogens such as Pseudomonas syringae pv. tomato DC3000, Xanthomonas vesicatoria, and Clavibacter michiganensis, as well as the fungal pathogen Botrytis cinerea . Unlike the 44 kDa chitinase that targets fungal cell walls, SlHBP2 appears to disrupt bacterial cell walls causing leakage of intracellular contents . Both proteins represent different aspects of the tomato's multilayered defense strategy against diverse pathogens.

What challenges exist in expressing recombinant tomato cell wall proteins in heterologous systems?

Recombinant expression of tomato cell wall proteins presents several significant challenges:

• Toxicity to host cells: Antimicrobial proteins like tomato chitinases may be toxic to the expression host. For example, Snakin-2 (SN2), another antimicrobial peptide from tomato, demonstrates toxicity against its producing E. coli strain, necessitating fusion with thioredoxin A to mitigate this effect .

• Disulfide bond formation: Many plant defense proteins, including chitinases, contain multiple disulfide bonds critical for their structure and function. Standard E. coli strains have a reducing cytoplasmic environment that inhibits proper disulfide bond formation .

• Post-translational modifications: Plant proteins often undergo glycosylation and other modifications that may not be properly replicated in prokaryotic expression systems, potentially affecting protein folding, stability, and activity .

• Codon usage bias: Differences in codon preference between plants and expression hosts may lead to poor translation efficiency, truncated products, or misfolding .

• Protein solubility: Cell wall proteins often contain hydrophobic regions that can lead to aggregation and inclusion body formation when expressed at high levels in heterologous systems .

These challenges require careful optimization of expression systems, fusion partners, and purification strategies to obtain functionally active recombinant proteins.

What expression systems are optimal for recombinant production of the 44 kDa tomato cell wall protein?

The selection of an appropriate expression system for the recombinant production of tomato cell wall proteins depends on several factors:

For the 44 kDa chitinase, a pragmatic approach might involve initial trials in specialized E. coli strains (such as SHuffle or Origami) with appropriate fusion partners (thioredoxin, MBP, or SUMO), followed by scale-up in more complex systems if necessary . The choice should be guided by the intended application of the recombinant protein and the required authenticity of its structural and functional properties.

How can we analyze the antimicrobial mechanism of action of the recombinant 44 kDa chitinase?

Understanding the antimicrobial mechanism of the 44 kDa chitinase requires a multi-faceted analytical approach:

• Enzymatic activity assays: Quantify chitinolytic activity using fluorogenic or chromogenic substrates to determine the hydrolytic potential against fungal cell wall components.

• Microscopy techniques: Employ fluorescence microscopy and electron microscopy to visualize the interaction between the recombinant protein and pathogen structures, as has been done with other antimicrobial proteins like SlHBP2 .

• Cell permeability assays: Measure the ability of the protein to cause membrane permeabilization and leakage of cellular contents from target pathogens using fluorescent dyes.

• Binding studies: Characterize substrate specificity and binding kinetics using surface plasmon resonance or isothermal titration calorimetry.

• Structure-function analysis: Correlate structural features with antimicrobial activity through site-directed mutagenesis of key residues.

• Synergy studies: Investigate potential synergistic effects with other tomato defense proteins, such as β-1,3-glucanases or thaumatin-like proteins that are co-expressed during pathogen attack .

• In vivo efficacy testing: Evaluate disease suppression in controlled plant infection assays using purified recombinant protein or transgenic expression.

These approaches would provide comprehensive insights into how the 44 kDa chitinase contributes to tomato defense and its potential applications in crop protection strategies.

What are the post-translational modifications of the native 44 kDa chitinase and how do they affect function?

Post-translational modifications (PTMs) can significantly impact the structure, stability, and function of plant defense proteins. For the 44 kDa chitinase from tomato, several potential PTMs may be critical:

• Phosphorylation: Proteomic analysis has identified significant changes in phosphorylation patterns of proteins during tomato fruit ripening and defense responses . Phosphorylation could regulate the activity or subcellular localization of the chitinase.

• Glycosylation: Many plant chitinases are N-glycosylated, which can affect protein stability, solubility, and resistance to proteolytic degradation. The specific glycosylation pattern may also influence interaction with target pathogens.

• Disulfide bond formation: Proper disulfide bonding is crucial for the structural integrity and catalytic activity of many chitinases. The search results indicate that some defense proteins from tomato, such as Snakin-2, contain multiple disulfide bonds that are essential for their antimicrobial function .

• Proteolytic processing: Some defense proteins require proteolytic cleavage of signal peptides or pro-domains for activation.

When producing recombinant versions of the 44 kDa chitinase, the choice of expression system should be guided by its ability to perform the necessary PTMs. For applications requiring native-like function, eukaryotic expression systems may be preferable despite their higher cost and complexity . Characterization of the native PTMs using mass spectrometry would be an important first step in designing an appropriate expression strategy.

How can we optimize yield and biological activity of the recombinant 44 kDa chitinase?

Optimizing the yield and biological activity of recombinant tomato 44 kDa chitinase requires a systematic approach addressing multiple aspects of protein expression and purification:

• Vector design optimization:

  • Codon optimization for the expression host

  • Selection of appropriate promoters (e.g., T7 for E. coli)

  • Inclusion of fusion tags that enhance solubility and facilitate purification

  • Incorporation of secretion signals if appropriate for the expression system

• Expression conditions:

  • Temperature reduction during induction (15-25°C) to facilitate proper folding

  • Optimized inducer concentration and induction timing

  • Supplementation with cofactors or stabilizing agents in the culture medium

  • Extension of expression time for higher yields

• Fusion partner selection:

  • Thioredoxin A has shown success with antimicrobial peptides from tomato

  • Maltose-binding protein (MBP) for enhanced solubility

  • SUMO tag for improved folding and optional native N-terminus after cleavage

• Purification strategy:

  • Multi-step chromatography approach beginning with affinity purification

  • Buffer optimization to maintain protein stability during purification

  • Careful removal of fusion tags while preserving activity

  • Limited exposure to harsh conditions that might denature the protein

• Activity preservation:

  • Addition of stabilizing agents (glycerol, specific ions) in storage buffers

  • Determination of optimal pH and temperature for storage

  • Lyophilization protocols for long-term stability if appropriate

Each of these factors should be systematically tested and optimized to achieve the highest yield of biologically active recombinant protein suitable for further research or applications.

What is the most effective protocol for purifying recombinant 44 kDa chitinase from E. coli?

Based on established methods for similar proteins, the following protocol is recommended for purifying recombinant tomato 44 kDa chitinase from E. coli:

• Cell lysis:

  • Harvest cells by centrifugation (6,000 × g, 15 min, 4°C)

  • Resuspend in lysis buffer containing appropriate protease inhibitors

  • Lyse cells by sonication or French press

  • Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Primary affinity purification:

  • Apply clarified lysate to an appropriate affinity column based on the fusion tag

  • For His-tagged constructs, use immobilized metal affinity chromatography (IMAC)

  • Wash extensively to remove non-specifically bound proteins

  • Elute with an imidazole gradient or specific competitive agents

• Tag removal (if necessary):

  • Dialyze against appropriate buffer for the specific protease

  • Add tag-specific protease (e.g., TEV, thrombin, or SUMO protease)

  • Incubate at optimal temperature (typically 4-16°C overnight)

  • Remove protease and cleaved tag by reverse affinity chromatography

• Secondary purification:

  • Ion exchange chromatography (e.g., DEAE-cellulose) to separate based on charge

  • Size exclusion chromatography (e.g., Sephacryl S-300) for final polishing

  • Consider hydroxyapatite chromatography for further purification if needed

• Quality control:

  • Verify purity by SDS-PAGE

  • Confirm identity by Western blot and mass spectrometry

  • Assess activity using appropriate enzymatic assays

  • Analyze protein homogeneity by dynamic light scattering

This protocol should be optimized based on the specific properties of the recombinant construct and the intended applications of the purified protein .

How can we assess the biological activity of purified recombinant chitinase?

Assessing the biological activity of purified recombinant 44 kDa chitinase requires a combination of biochemical and microbiological approaches:

• Enzymatic activity assays:

  • Colorimetric assay using chromogenic substrates (e.g., 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside)

  • Turbidimetric assay measuring clearing of colloidal chitin suspensions

  • Viscometric analysis monitoring the decrease in viscosity of chitin solutions

  • Reducing sugar assays to quantify released N-acetylglucosamine

• Antimicrobial activity testing:

  • Microdilution assays to determine minimum inhibitory concentrations (MICs) against relevant fungal pathogens

  • Disk diffusion assays to visualize zones of inhibition

  • Time-kill kinetics to establish the rate of antimicrobial action

  • Synergy studies with other defense proteins (e.g., β-1,3-glucanases)

• Structural integrity assessment:

  • Circular dichroism spectroscopy to confirm proper secondary structure

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to probe correct folding

• Binding studies:

  • Surface plasmon resonance to measure binding kinetics to chitin substrates

  • Affinity electrophoresis using chitin-containing matrices

  • Fluorescence-based interaction assays with labeled substrates

• In planta assays:

  • Detached leaf assays treating leaves with purified protein before pathogen challenge

  • Disease symptom reduction measurements in greenhouse trials

  • Microscopic examination of pathogen structures after protein treatment

These complementary approaches provide a comprehensive assessment of both the enzymatic and antimicrobial properties of the recombinant chitinase .

What techniques are available for analyzing the structure-function relationship of the 44 kDa chitinase?

Understanding the structure-function relationship of the tomato 44 kDa chitinase requires integrating multiple analytical techniques:

• High-resolution structural analysis:

  • X-ray crystallography to determine three-dimensional structure at atomic resolution

  • Nuclear magnetic resonance (NMR) spectroscopy for solution structure and dynamics

  • Cryo-electron microscopy for visualizing larger complexes with substrates or partners

• Computational approaches:

  • Homology modeling based on related chitinases with known structures

  • Molecular dynamics simulations to study substrate binding and catalytic mechanisms

  • Docking studies to predict interactions with fungal cell wall components

• Mutational analysis:

  • Alanine scanning of key residues to identify those critical for activity

  • Site-directed mutagenesis targeting the catalytic site and substrate-binding regions

  • Domain swapping with other chitinases to create chimeric proteins

• Spectroscopic methods:

  • Circular dichroism to monitor secondary structure changes upon substrate binding

  • Fluorescence spectroscopy to detect conformational changes

  • Fourier-transform infrared spectroscopy to analyze specific structural elements

• Interaction studies:

  • Surface plasmon resonance (SPR) to measure binding kinetics with substrates

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Hydrogen-deuterium exchange mass spectrometry to identify regions involved in binding

• Functional correlation:

  • Systematic comparison of structure-based variants with enzymatic and antimicrobial activities

  • Analysis of the effects of post-translational modifications on structure and function

  • Correlation of thermal stability with functional parameters

These approaches would provide insights into the molecular basis of the chitinase's activity and guide protein engineering efforts to enhance its antimicrobial properties .

What are the key considerations for scaling up production of recombinant tomato cell wall proteins?

Scaling up the production of recombinant tomato 44 kDa chitinase from laboratory to larger scales requires addressing several critical factors:

• Fermentation optimization:

  • Selection of appropriate media formulation (defined vs. complex)

  • Feeding strategies for high-density cultivation

  • Optimization of dissolved oxygen levels and pH control

  • Temperature control, especially during induction phases

  • Determination of optimal induction parameters at scale

• Process considerations:

  • Consistent cell lysis methods suitable for large volumes

  • Filtration strategies to efficiently clarify large volumes of lysate

  • Chromatography approaches amenable to scale-up (larger columns, faster flow rates)

  • Process analytical technology (PAT) implementation for real-time monitoring

• Protein stability factors:

  • Buffer formulations that enhance stability during processing and storage

  • Minimization of proteolytic degradation through inhibitor addition or host strain selection

  • Assessment of temperature sensitivity during processing steps

  • Evaluation of freeze-thaw stability for intermediate storage

• Quality control:

  • Development of robust analytical methods for in-process testing

  • Implementation of consistent lot release criteria

  • Stability-indicating assays to monitor activity retention

  • Contaminant profile analysis, particularly for endotoxin levels

• Cost efficiency:

  • Optimization of yield per unit volume to reduce costs

  • Reusability of chromatography resins and other materials

  • Energy consumption reduction strategies

  • Waste management and environmental considerations

• Regulatory considerations (if applicable):

  • Consistent documentation of process parameters

  • Validation of purification steps for reproducibility

  • Establishment of specifications for final product quality

By systematically addressing these considerations, researchers can develop a scalable and cost-effective process for producing the recombinant 44 kDa chitinase at quantities sufficient for research or potential agricultural applications .