Recombinant Cynara cardunculus Cardosin-F, partial
Description
Recombinant Cardosin B Production in Tobacco BY2 Cells
Tobacco BY2 cell cultures were engineered to express recombinant cardosin B, achieving intracellular retention of the enzyme. Key outcomes include:
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Localization: Unlike native cardosin B (secreted in cardoon flowers), the recombinant form remained intracellular in BY2 cells, detected exclusively in cellular extracts via western blot .
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Glycosylation: Endoglycosidase H assays confirmed high-mannose glycosylation, consistent with patterns observed in native cardoon flowers .
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Yield: A purification pipeline using Q-sepharose anion exchange chromatography enabled isolation of active cardosin B with a single chromatographic step, achieving >90% purity .
Protein Processing and Activation
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Unprocessed Form Detection: Fusion of cardosin B with dsRed fluorescent protein delayed processing, revealing an unprocessed 75–100 kDa form. This intermediate was purified via immobilized metal affinity chromatography (IMAC) and shown to activate under acidic conditions (pH 4) .
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Proteolytic Activation: Acidic incubation reduced the unprocessed form to the mature 34 kDa heavy chain. Pepstatin A (an AP inhibitor) partially blocked activation, while combined use with E64 (cysteine protease inhibitor) fully inhibited processing, implicating cysteine proteases in maturation .
Enzymatic Activity
Interactions with Cysteine Proteases
Mass spectrometry identified three cysteine proteases in BY2 cells that may regulate cardosin B processing:
| Protease ID | Characteristics | Role in Cardosin B Processing |
|---|---|---|
| CP15 | 97% identity to low-temperature induced protease | Potential cleavage of propeptide |
| CP6 | 77% identity to low-temperature induced protease | Facilitates maturation |
| Granulin-domain protease | Papain-like with unknown function | Suspected interaction with saposin-like domain of cardosin B |
These proteases contain granulin-domains, which may mediate interactions with cardosin B’s saposin-like domain, analogous to human progranulin-cathepsin D interactions .
Challenges and Industrial Relevance
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Heterogeneity: Native cardoon APs exhibit variability in milk clotting activity, complicating standardization .
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Scalability: BY2 cells offer a stable platform for recombinant cardosin B production, though yields remain lower than microbial systems .
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Applications: Recombinant cardosin B retains milk clotting functionality, making it a viable alternative to animal-derived rennet for vegetarian cheese production .
Future Directions
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Trafficking Studies: Confocal microscopy hints at vacuolar sorting of cardosin B-dsRed fusion proteins, warranting deeper investigation into secretory pathways .
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Process Optimization: Enhancing yield via culture condition adjustments or genetic engineering of BY2 cells.
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Structural Studies: Resolving the 3D structure of recombinant cardosin B to refine its industrial application.
Product Specs
Target Background
Q&A
What expression systems are optimal for producing recombinant Cardosin-F (partial) with retained enzymatic activity?
To achieve functional Cardosin-F, prokaryotic (e.g., E. coli BL21) and eukaryotic (e.g., Pichia pastoris) systems are commonly compared. E. coli often requires codon optimization and refolding protocols due to insolubility, while P. pastoris enables proper post-translational modifications. For example, cardosin B expressed in E. coli showed 40% solubility with a 0.39 U/mL milk-clotting activity after refolding . Key steps:
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Use pET or pPICZα vectors for cloning.
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Induce with 0.5 mM IPTG (prokaryotic) or 1% methanol (eukaryotic).
Table 1: Expression Systems for Cardosin-F Variants
| System | Solubility (%) | Specific Activity (U/mg) | Refolding Required |
|---|---|---|---|
| E. coli BL21 | 40–60 | 0.39–1.2 | Yes |
| P. pastoris | >80 | 2.5–3.8 | No |
| Arabidopsis | 95 | 4.1 | No |
Data derived from heterologous expression of cardosin A/B .
How can researchers purify recombinant Cardosin-F (partial) to >90% homogeneity?
A three-step protocol is recommended:
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Affinity chromatography: Use His-tag purification with Ni-NTA resin (pH 7.4).
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Ion-exchange chromatography: Apply a 0–1 M NaCl gradient (pH 5.0–6.0) to resolve isoforms.
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Size-exclusion chromatography: Remove aggregates using Superdex 200 .
Cardosin B purification achieved 92% purity with a 15% yield using this workflow .
What assays reliably measure Cardosin-F’s enzymatic activity and substrate specificity?
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Caseinolytic assay: Monitor absorbance at 280 nm after 30 min incubation (pH 5.0, 37°C) .
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SDS-PAGE zymography: Resolve proteins on 12% gels copolymerized with 0.1% gelatin.
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HPLC peptide profiling: Hydrolyze β-lactoglobulin (β-Lg) and quantify cleaved peptides .
Cardosin B hydrolyzed β-Lg by ~40% at pH 6.29/50°C, while cardosin A showed broader specificity .
How can solubility issues during recombinant expression be systematically resolved?
Experimental Design:
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Screen solubility tags: Test MBP, GST, or SUMO fusions.
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Optimize induction: Lower temperature (18°C) and IPTG concentration (0.1 mM).
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Refolding gradient: Use urea (0–6 M) in 50 mM Tris (pH 8.0) with 5 mM reduced glutathione .
In cardosin B, solubilization with 4 M urea and stepwise dialysis restored 70% activity .
What structural motifs dictate Cardosin-F’s substrate specificity and pH dependence?
Cardosin-F’s specificity arises from:
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Catalytic dyad: Asp32 and Asp215 (numbered via homology to cardosin A) .
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Hydrophobic S1 pocket: Binds aromatic residues (e.g., Phe in κ-casein).
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N-terminal prodomain: Autoprocessed at pH < 5.0 to activate the enzyme .
Deletion of the plant-specific insert (PSI) in cardosin A abolished collagenolytic activity .
Table 2: Functional Domains in Cardosin-F Homologs
| Domain | Function | pH Sensitivity |
|---|---|---|
| Prodomain | Auto-inhibition | <5.0 |
| Catalytic domain | Substrate cleavage | 4.5–6.5 |
| PSI | Membrane interaction | N/A |
Adapted from cardosin A/B structural studies .
How do conflicting data on Cardosin-F’s β-Lg hydrolysis arise, and how can they be reconciled?
Discrepancies stem from:
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Protease isoforms: Cardosin A (broad specificity) vs. Cardosin B (narrow specificity) .
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Assay conditions: DH values for β-Lg peaked at pH 6.29/50°C but were undetectable at pH 5.01/64°C .
Resolution: -
Standardize substrate ratios (E/S = 1:150 w/w).
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Pre-incubate β-Lg at 65°C for 15 min to expose cleavage sites .
What in vitro models validate Cardosin-F’s biological roles beyond enzymatic activity?
Integrin-mediated endocytosis assay:
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Incubate A549 cells with Alexa488-labeled Cardosin-F (10°C, 1 hr).
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Block integrin β1 with 10 µM RGDS peptide.
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Quantify internalization via flow cytometry .
Cardosin A showed 60% reduced uptake upon integrin inhibition, confirming receptor-mediated entry .
How can computational tools predict Cardosin-F’s cleavage sites in complex substrates?
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PeptideCutter: Input substrate sequences (e.g., α-lactalbumin) with pH/temperature constraints.
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MD simulations: Model substrate docking using ROSETTA.
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Machine learning: Train on cardosin A/B cleavage data (e.g., P₁ preference for Phe/Leu) .
What strategies confirm the absence of endotoxins in therapeutic-grade Cardosin-F?
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Limulus Amebocyte Lysate (LAL) assay: Detect endotoxins at <0.1 EU/mg.
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Ion-exchange polishing: Use Q Sepharose at pH 8.0.
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FTIR spectroscopy: Identify lipid A contaminants (peak at 1,740 cm⁻¹) .
Methodological Case Study: Optimizing Hydrolysis Conditions
Central Composite Design (CCD) for DH maximization:
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Variables: pH (5.0–7.0), temperature (35–65°C).
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Response: Degree of hydrolysis (DH), ACE-inhibitory activity .
Table 3: CCD Results for β-Lg Hydrolysis
| pH | Temp (°C) | DH (%) | ACE Inhibition (%) |
|---|---|---|---|
| 5.01 | 64.14 | 0 | 0 |
| 6.29 | 50.00 | 40.2 | 78.5 |
| 6.99 | 35.86 | 100 | 92.3 |
Optimal conditions: pH 6.29/50°C for balanced DH and bioactivity .
