Recombinant Solanum lycopersicum 40 kDa cell wall protein

Basic Research Questions

• What is the Solanum lycopersicum 40 kDa cell wall protein?

  The 40 kDa cell wall protein from Solanum lycopersicum (tomato) has been identified as chlorogenate:glucarate caffeoyltransferase (SlCGT), an enzyme involved in cell wall metabolism. This protein was isolated from tomato seedlings and characterized through various biochemical approaches. SlCGT is part of the broader family of acyltransferases and plays roles in cell wall development. The protein has been confirmed to have a molecular weight of approximately 40 kDa when analyzed by SDS-PAGE, and its sequence has been determined through mass spectrometric sequencing of purified protein from tomato seedlings .

• Where is the 40 kDa cell wall protein localized in tomato plants?

  Subcellular localization studies using immunogold electron microscopy with SlCGT-specific antibodies have demonstrated that the 40 kDa cell wall protein is primarily localized in the cell wall and associated structures. The localization was determined by generating SlCGT-specific antibodies by immunizing rabbits with purified SlCGT from 7-day-old tomato seedlings, followed by affinity chromatography enrichment using protein A-Sepharose. For visualization, ultrathin sections mounted on copper grids were treated with the anti-SlCGT antibody and protein A conjugated with 10 nm colloidal gold, then poststained with uranyl acetate and lead citrate before examination with a Philips CM 10 electron microscope .

• How can I isolate the native 40 kDa cell wall protein from tomato tissue?

  Isolation of the native SlCGT from tomato tissue follows a multi-step purification process:

  1. Prepare crude extracts from tomato seedlings

  2. Perform ammonium sulfate precipitation

  3. Dissolve the precipitate in 0.02 M Tris buffer (pH 7.0) with 1 M ammonium sulfate

  4. Fractionate using phenyl-Sepharose 16/10 column chromatography with a gradient of 0.02 M Tris buffer and 1 M ammonium sulfate

  5. Pool active fractions and concentrate by ultrafiltration

  6. Further purify using Superdex G-75 16/60 size exclusion chromatography with 0.01 M citrate buffer (pH 5)

  7. Perform final purification using Mono Q anion exchange chromatography

  Protein concentrations should be determined during all fractionation steps, and activity assays should be performed to track the enzyme through the purification process .

Intermediate Research Questions

• What methods are most effective for cloning and expressing the recombinant 40 kDa cell wall protein?

  For successful cloning and expression:

  1. RNA Extraction and cDNA Synthesis: Extract total RNA from 5-day-old tomato seedlings and enrich for poly(A) RNA for cDNA synthesis.

  2. Gene Amplification: Amplify the full-length cDNA using PCR with specific primers designed from the SlCGT sequence.

  3. Expression Vector Selection: Clone the SlCGT cDNA into an expression vector such as pImpact1.1 under the control of an appropriate promoter (e.g., rbcs promoter from Asteraceous chrysanthemum).

  4. Expression System: Transfer the expression cassette to a binary vector like pBINPLUS for transformation into Agrobacterium tumefaciens strain GV2260.

  5. Transient Expression: Perform transient expression in Nicotiana benthamiana leaves through Agrobacterium infiltration. Include controls such as empty vector and a GUS gene construct to normalize transformation efficiency.

  6. Protein Extraction: Extract soluble proteins from transformed leaves, desalt using a PD-10 column, and concentrate for activity assays .

  For higher yields, consider the fractional experimental design approach used for plant cell wall glycosyltransferases, which systematically tests factors influencing recombinant protein expression .

• How can I optimize solubility of the recombinant 40 kDa cell wall protein during expression?

  Research on plant cell wall glycosyltransferases has shown that these proteins generally have a very low soluble:insoluble ratio in heterologous expression systems. To improve solubility:

  1. Co-expression with Chaperones: Co-express molecular chaperones to assist in proper protein folding

  2. Lysis Buffer Optimization: Optimize lysis buffer composition to enhance protein solubility during extraction

  3. Expression Conditions: Modify temperature, induction time, and inducer concentration to favor soluble protein production

  4. Fusion Tags: Test different solubility-enhancing fusion tags, such as MBP, SUMO, or thioredoxin

  5. Host Selection: Screen multiple expression hosts, as some plant proteins show better solubility in specific systems

  A high-throughput screening approach can be used to identify optimal conditions for soluble expression before scaling up production .

• What are the most reliable methods for confirming the identity and purity of the recombinant protein?

  To confirm identity and purity:

  1. SDS-PAGE: Analyze protein size and preliminary purity assessment

  2. Western Blotting: Use SlCGT-specific antibodies generated against the purified native protein

  3. Mass Spectrometry: Perform tryptic digestion followed by LC-MS/MS analysis to confirm identity through peptide matching

  4. Activity Assays: Measure enzymatic activity using specific substrates

  5. Size Exclusion Chromatography: Assess protein homogeneity and detect aggregation

  6. N-terminal Sequencing: Confirm the correct processing of the N-terminus

  For SlCGT, immunological detection using specific antibodies at 1:500 dilution followed by a secondary goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (1:1000) has been effective .

Advanced Research Questions

• How can site-directed mutagenesis be used to identify catalytic residues in the 40 kDa cell wall protein?

  Site-directed mutagenesis can be performed using the following methodology:

  1. Prediction of Catalytic Residues: Analyze sequence alignments with related proteins to identify conserved amino acids potentially involved in catalysis

  2. Mutagenesis Protocol: Use the QuikChange XL Site-directed Mutagenesis kit or similar system to introduce specific mutations

  3. Verification: Confirm all mutations by DNA sequencing

  4. Expression System: Express wild-type and mutant variants in N. benthamiana using the Agrobacterium-mediated transient expression system

  5. Activity Analysis: Compare enzyme activities between wild-type and mutant proteins to identify essential catalytic residues

  6. Structure-Function Correlation: Correlate the activity data with structural models to understand the role of specific residues

  For SlCGT, control experiments should include co-transformation with a GUS construct to normalize for transformation efficiency across experiments .

• What strategies are effective for structural characterization of the recombinant 40 kDa cell wall protein?

  For structural characterization:

  1. Protein Preparation: Purify the protein to >95% homogeneity with yields sufficient for structural studies (typically several milligrams)

  2. Circular Dichroism (CD): Analyze secondary structure content and thermal stability

  3. Limited Proteolysis: Identify stable domains for crystallization attempts

  4. Crystallization Screens: Perform high-throughput crystallization screening with various precipitants, buffers, and additives

  5. X-ray Crystallography: Collect diffraction data and solve structure

  6. NMR Spectroscopy: For analysis of protein dynamics and ligand interactions

  7. Cryo-EM: Alternative approach for structural determination if crystallization proves challenging

  8. In Silico Modeling: Generate homology models based on related structures while experimental structures are being determined

  The high-throughput approaches used for plant cell wall glycosyltransferases could be adapted for structural studies of SlCGT .

• How does the recombinant 40 kDa cell wall protein compare functionally to the native protein?

  To compare recombinant and native proteins:

  1. Enzymatic Activity: Measure specific activity, substrate specificity, and kinetic parameters (Km, Vmax, kcat)

  2. Post-translational Modifications: Analyze glycosylation, phosphorylation, and other modifications using mass spectrometry

  3. Thermal Stability: Compare thermal denaturation profiles using differential scanning fluorimetry

  4. pH and Temperature Optima: Determine and compare optimal conditions for enzyme activity

  5. Protein-Protein Interactions: Assess binding to known interaction partners

  6. Inhibitor Sensitivity: Compare response to known inhibitors

  For SlCGT, activity assays should be standardized using internal controls to enable direct comparison between native and recombinant forms .

• What are the challenges and solutions for scaling up production of recombinant Solanum lycopersicum 40 kDa cell wall protein?

  Challenges and solutions for scale-up:

  Challenges:

  • Low soluble:insoluble ratio common in plant cell wall proteins

  • Potential toxicity to expression hosts

  • Loss of activity during purification

  • Protein instability during storage

  Solutions:

  • Expression System Optimization: Test multiple expression systems including bacterial, yeast, insect, and plant-based platforms

  • Bioreactor Cultivation: Implement controlled bioreactor conditions for optimal growth and expression

  • Fusion Protein Strategies: Use solubility-enhancing tags with efficient removal systems

  • Purification Train Development: Design multi-step purification processes to maintain activity

  • Stabilization Formulations: Develop buffer compositions with appropriate additives to enhance stability

  • Storage Conditions: Optimize freezing/lyophilization protocols to maintain activity

  High-throughput screening methods can identify improved conditions before implementing larger-scale production .

• How can genome editing be used to study the function of the 40 kDa cell wall protein in planta?

  CRISPR/Cas9-based genome editing approach:

  1. Target Site Selection: Identify suitable target sites in the SlCGT gene using CRISPR design tools

  2. Guide RNA Design: Design specific guide RNAs targeting conserved regions of the gene

  3. Vector Construction: Clone guide RNAs into a CRISPR/Cas9 expression vector suitable for plant transformation

  4. Transformation: Transform tomato plants using Agrobacterium-mediated methods

  5. Mutant Screening: Screen transformants for mutations using PCR-based genotyping and sequencing

  6. Phenotypic Analysis: Evaluate mutant plants for:

     • Cell wall composition changes

     • Altered growth and development

     • Response to biotic and abiotic stresses

     • Changes in cell wall-related enzyme activities

  7. Complementation Studies: Confirm phenotypes by complementing mutants with the wild-type gene

  8. Transcriptomic Analysis: Perform RNA-seq to identify changes in gene expression networks

• What techniques are available for studying the interaction of the 40 kDa cell wall protein with other cell wall components?

  To study protein-cell wall interactions:

  1. Affinity Purification: Use tagged versions of the protein to identify interacting partners

  2. Surface Plasmon Resonance (SPR): Quantify binding kinetics to purified cell wall components

  3. Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of binding

  4. Bimolecular Fluorescence Complementation (BiFC): Visualize protein interactions in planta

  5. Fluorescence Resonance Energy Transfer (FRET): Study protein proximity in real-time

  6. Co-immunoprecipitation: Identify protein complexes using antibodies against SlCGT

  7. Crosslinking Mass Spectrometry: Identify proximity relationships between proteins

  8. Solid-state NMR: Study interactions with insoluble cell wall components

  Immunogold electron microscopy, as used for SlCGT localization, can also reveal co-localization with other cell wall components .