CASP3 Human, Sf9
Description
Production and Purification
The protein is produced in Sf9 cells infected with recombinant baculovirus, followed by proprietary chromatographic purification .
| Parameter | Details |
|---|---|
| Expression System | Sf9 Baculovirus cells |
| Purification Method | Nickel-affinity chromatography |
| Formulation | 20 mM HEPES (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 20% glycerol, 1 mM DTT |
| Storage | Short-term: 4°C; Long-term: -20°C with carrier protein (e.g., 0.1% HSA/BSA) |
Enzymatic Activity
CASP3 Human, Sf9 exhibits robust caspase-3-like activity, cleaving synthetic substrates (e.g., DEVD-AFC) and physiological targets (e.g., pro-caspase-3, PARP1) .
Activity Data:
| Substrate | Activity | Km (Pro-Caspase-3) |
|---|---|---|
| DEVD-AFC | >5,000 pmol/min/μg | — |
| Pro-Caspase-3 | Facilitates apoptosome-mediated activation | 139.3 ± 23.19 nM |
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Optimal pH: 5.5 for recombinant AtCathB3 (a plant homolog with caspase-3-like activity) .
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Inhibition: Suppressed by caspase-3 inhibitors (e.g., Ac-DEVD-CHO) .
Apoptosis Execution:
Non-Apoptotic Functions:
Research Applications:
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Drug Discovery: Screening for caspase-3 inhibitors.
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Biochemical Assays: Quantifying apoptosis in cell lysates (e.g., via ELISA) .
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Structural Studies: Investigating caspase activation mechanisms .
Comparative Insights
CASP3 Human, Sf9 shares functional parallels with plant cathepsin B3 (AtCathB3), which exhibits caspase-3-like activity and regulates programmed cell death (PCD) in Arabidopsis . Both enzymes are inhibited by synthetic caspase-3 inhibitors, underscoring evolutionary conservation in PCD pathways .
Technical Considerations
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Stability: Maintain activity by avoiding freeze-thaw cycles; add carrier proteins for long-term storage .
Emerging Research Directions
Product Specs
Caspase 3, also known as Apoptosis-Related Cysteine Peptidase (CASP3), is a protein belonging to the cysteine-aspartic acid protease (caspase) family. These proteases play a crucial role in the execution phase of programmed cell death, or apoptosis. Caspases are initially present in cells as inactive precursors called proenzymes. Upon receiving specific signals, these proenzymes undergo a series of cleavages at conserved aspartic acid residues. This processing generates two subunits, a large and a small subunit, which then combine to form the active caspase enzyme. CASP3, once activated, can further activate other caspases, including caspases 6, 7, and 9, amplifying the apoptotic cascade. Conversely, CASP3 itself can be activated by caspases 8, 9, and 10. This intricate activation network highlights the tightly regulated nature of apoptosis. CASP3 has been implicated in several key cellular processes, including: 1. Cleavage of Amyloid-beta 4A Precursor Protein: CASP3 is a major enzyme responsible for cleaving the amyloid-beta 4A precursor protein, a process linked to neuronal death in Alzheimer's disease. 2. Cleavage of Huntingtin: CASP3 is involved in the breakdown of huntingtin, a protein whose mutation is associated with Huntington's disease. 3. Cleavage of Sterol Regulatory Element Binding Proteins (SREBPs): CASP3 can cleave SREBPs, transcription factors involved in lipid metabolism, separating their DNA-binding domain from the membrane attachment domain. 4. Initiation of Cell Adhesion: In sympathetic neurons, CASP3 plays a role in initiating cell adhesion by cleaving the RET receptor tyrosine kinase.
This product consists of the human CASP3 protein produced using Sf9 insect cells infected with a baculovirus expression system. It is a single polypeptide chain that has undergone glycosylation, a common post-translational modification. The protein encompasses amino acids 29 to 277 of the CASP3 sequence, resulting in a protein of 256 amino acids. The molecular weight of the protein is 29.4 kDa. On SDS-PAGE analysis under reducing conditions, it migrates with an apparent molecular weight between 13.5 and 18 kDa. The protein has been engineered to include a 6-amino acid histidine tag at the C-terminus, facilitating its purification. Purification is achieved using proprietary chromatographic techniques.
The CASP3 protein is provided in a solution at a concentration of 0.5 mg/ml. The solution is buffered with 20 mM HEPES at pH 7.5 and contains 0.1 M NaCl, 1 mM EDTA, 20% glycerol, and 1 mM DTT.
For short-term storage (up to four weeks), the CASP3 protein solution can be stored at 4°C. For extended storage, it is recommended to store the protein at -20°C. To ensure optimal stability during long-term storage, consider adding a carrier protein such as albumin (HSA or BSA) to a final concentration of 0.1%. It is crucial to minimize freeze-thaw cycles to maintain protein integrity.
The purity of the CASP3 protein is greater than 90%, as determined by SDS-PAGE analysis.
The specific activity of the CASP3 protein is greater than 5,000 pmol/min/µg. This activity is determined by measuring the enzyme's ability to cleave a specific substrate, Ac-DEVD-AFC, into Ac-DEVD and AFC. One unit of enzyme activity is defined as the amount of enzyme required to release 1 pmole of AFC per minute at a pH of 7.5 and a temperature of 25°C.
CASP3, CPP32, CPP32B, SCA-1, CASP-3, Apopain, Cysteine protease CPP32, CPP-32, Protein Yama, SREBP cleavage activity 1.
MSGISLDNSY KMDYPEMGLC IIINNKNFHK STGMTSRSGT DVDAANLRET FRNLKYEVRN
KNDLTREEIV ELMRDVSKED HSKRSSFVCV LLSHGEEGII FGTNGPVDLK KITNFFRGDR
CRSLTGKPKL FIIQACRGTE LDCGIETDSG VDDDMACHKI PVEADFLYAY STAPGYYSWR
NSKDGSWFIQ SLCAMLKQYA DKLEFMHILT RVNRKVATEF ESFSFDATFH AKKQIPCIVS MLTKELYFYH HHHHHH
Q&A
What is CASP3 Human, Sf9?
CASP3 Human, Sf9 is a recombinant human Caspase-3 protein expressed in Spodoptera frugiperda 9 (Sf9) insect cells using a baculovirus expression system. It is produced as a single glycosylated polypeptide chain containing 256 amino acids (29-277 a.a.) with a molecular mass of 29.4kDa, though it migrates at 13.5-18kDa on SDS-PAGE under reducing conditions. The protein is expressed with a 6 amino acid His tag at the C-terminus and is purified using proprietary chromatographic techniques to achieve greater than 90% purity .
What is the role of CASP3 in cellular pathways?
CASP3 belongs to the cysteine-aspartic acid protease (caspase) family and plays a key role in the execution phase of cell apoptosis. It exists as an inactive proenzyme that undergoes proteolytic processing at conserved aspartic residues to generate two subunits (large and small) that dimerize to form the active enzyme. Once activated, CASP3 cleaves and activates caspases 6, 7, and 9, while it is itself processed by caspases 8, 9, and 10. CASP3 is the primary caspase involved in the cleavage of amyloid-beta 4A precursor protein (linked to neuronal death in Alzheimer's disease), huntingtin, and sterol regulatory element binding proteins (SREBPs) .
What are the optimal storage conditions for CASP3 Human, Sf9?
For short-term use (2-4 weeks), CASP3 Human, Sf9 can be stored at 4°C. For longer periods, storage at -20°C is recommended. The protein is typically supplied as a 0.5mg/ml solution containing 20mM HEPES buffer (pH 7.5), 0.1M NaCl, 1mM EDTA, 20% Glycerol, and 1mM DTT. For extended storage, addition of a carrier protein (0.1% HSA or BSA) is recommended to maintain stability. Multiple freeze-thaw cycles should be strictly avoided as they can compromise protein activity and structural integrity .
What controls should be included in CASP3 activity assays?
When designing experiments to measure CASP3 activity, researchers should include:
| Control Type | Purpose | Example |
|---|---|---|
| Positive control | Confirm assay functionality | Cells treated with known apoptosis inducer |
| Negative control | Establish baseline | Untreated cells |
| Inhibitor control | Verify specificity | Cells with caspase-3 inhibitor (e.g., Z-DEVD-FMK) |
| Time course | Track activation kinetics | Samples at multiple timepoints post-treatment |
| Technical replicates | Ensure reproducibility | Minimum three per condition |
Additional controls should be specific to the experimental context, such as transfection controls when gene manipulation is involved .
How can researchers effectively transfect Sf9 cells for CASP3 studies?
Effective transfection of Sf9 cells requires optimization of several parameters. For siRNA or antisense oligonucleotides (e.g., AS-miR-31), typically 0.50 μg combined with an appropriate transfection reagent like RNAiFect in a 1:5 ratio (w/v) is effective. For plasmid transfection (e.g., p53 expression vectors), 1 μg of DNA with 6 μl of a reagent like SuperFect has been successfully used. Transfections should be performed when cells are in the exponential growth phase, and expression analysis should be conducted at appropriate timepoints (16-24 hours for RNA interference, 24-48 hours for protein expression) .
How does p53 modulate CASP3 activation in response to radiation?
Research has uncovered a fascinating regulatory mechanism involving p53, miR-31, and CASP3 in radiation response. At sub-lethal radiation doses, Sf9 cells exhibit hyper-phosphorylation of p53, which then binds near the miR-31 genomic locus and suppresses its expression. This suppression of miR-31 prevents CASP3 activation and subsequent apoptosis. In contrast, at lethal radiation doses, p53 phosphorylation decreases, allowing miR-31 expression to increase, which leads to CASP3 activation and apoptotic cell death. This represents a unique radiation-responsive 'p53 gateway' that regulates apoptosis .
What is the significance of miR-31 in regulating CASP3-mediated apoptosis?
miR-31 appears to be a critical mediator in the radiation-induced apoptotic pathway. Research indicates an inverse relationship between radiation dose, miR-31 expression, and CASP3 activity. At sub-lethal doses, miR-31 is downregulated through p53-mediated suppression, correlating with reduced CASP3 activity and cell survival. At lethal doses, miR-31 is upregulated, leading to increased CASP3 activity and apoptotic cell death. This pathway represents a potential target for modulating radiation sensitivity in various applications .
How can findings from Sf9 cell studies on CASP3 be translated to human systems?
Translating findings from Sf9 cells to human systems requires consideration of both conserved elements and species-specific differences:
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Focus on evolutionarily conserved aspects of apoptosis mechanisms
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Assess structural and functional similarities between insect and human proteins
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Confirm key findings in human cell lines or animal models
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Adjust experimental parameters (e.g., radiation doses) appropriately for different model systems
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Map detailed signaling pathways in both systems to identify points of convergence and divergence
The study of CASP3 in Sf9 cells has particular relevance as Sfp53 (p53 in Sf9 cells) shows good functional homology with human p53, suggesting that the p53-miR-31-CASP3 pathway may have important implications for understanding and modulating human cell radioresistance .
What factors might affect the reliability of CASP3 activity measurements?
Several factors can influence CASP3 activity measurements and potentially lead to variability or inconsistent results:
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Sample preparation: Protein degradation during cell lysis can affect results
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Timing of measurement: CASP3 activation can be transient and time-dependent
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Cell culture conditions: Density, passage number, and growth phase affect baseline activity
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Reagent quality: Substrate specificity and detection sensitivity vary between assay kits
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Treatment parameters: Concentration, duration, and delivery method of apoptotic stimuli
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Cell heterogeneity: Subpopulations within cultures may have different activation thresholds
To minimize variability, researchers should standardize protocols, include appropriate controls, and perform sufficient biological and technical replicates .
How can researchers distinguish between active and inactive forms of CASP3?
Distinguishing between the inactive proenzyme (procaspase-3) and the active form of CASP3 requires specific methodological approaches:
| Method | Principle | Advantages | Limitations |
|---|---|---|---|
| Western blotting | Detects both full-length (32kDa) and cleaved fragments (17-19kDa) | Visual confirmation of processing | Semi-quantitative, requires specific antibodies |
| Fluorogenic substrate assay | Measures enzymatic activity using specific substrates (e.g., DEVD-AMC) | Quantitative, sensitive | Potential cross-reactivity with other caspases |
| Immunocytochemistry | Localizes active caspase in cellular compartments | Provides spatial information | Requires cell fixation, potential artifacts |
| Flow cytometry | Measures activity in individual cells | Single-cell resolution, statistical power | Requires specialized equipment |
For comprehensive analysis, combining multiple methods provides the most reliable results .
What experimental approaches can resolve contradictory CASP3 activation data?
When faced with contradictory results in CASP3 activation studies, researchers should consider:
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Temporal dynamics: Perform detailed time course experiments to capture activation kinetics
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Dose response: Establish comprehensive dose-response curves to identify threshold effects
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Cell-specific factors: Test multiple cell lines or primary cells to account for cell type variation
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Pathway analysis: Examine upstream regulators and downstream targets to identify points of divergence
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Technical validation: Verify reagent quality, instrument calibration, and methodology
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Complementary assays: Use orthogonal methods to confirm apoptosis (DNA fragmentation, membrane changes)
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Single-cell analysis: Consider heterogeneous responses that might be masked in population studies
This systematic approach can help reconcile seemingly contradictory findings and provide a more complete understanding of CASP3 regulation .
How can the p53-miR-31-CASP3 pathway inform radiation protection strategies?
The discovery of the p53-miR-31-CASP3 regulatory pathway reveals potential targets for radiation protection strategies. Research demonstrates that sub-lethal radiation causes hyper-phosphorylation of p53, which suppresses miR-31 and prevents CASP3 activation, thereby protecting cells from subsequent lethal radiation. This suggests several potential applications:
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Development of pharmacological agents that induce p53 phosphorylation without DNA damage
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miR-31 inhibitors that could mimic the protective effect of hyper-phosphorylated p53
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Pre-conditioning protocols that activate this protective pathway before anticipated radiation exposure
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Biomarkers to predict individual radiation sensitivity based on p53 phosphorylation capacity
These approaches could have significant implications for radiation protection in occupational, medical, and space exploration settings .
What methodological approaches can best characterize the CASP3 interactome?
Understanding the complete network of CASP3 interactions requires multi-faceted approaches:
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Proteomics: Mass spectrometry-based identification of binding partners
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Co-immunoprecipitation: Validation of specific protein-protein interactions
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Proximity labeling: Identification of transient or weak interactions in living cells
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Yeast two-hybrid screening: Systematic identification of potential binding partners
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Structural biology: Crystallography or cryo-EM to reveal binding interfaces
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Functional genomics: CRISPR screens to identify genetic modulators of CASP3 activity
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Computational modeling: Prediction of interaction networks based on structural and functional data
Integration of these approaches provides a comprehensive view of the CASP3 interactome in different cellular contexts and in response to various stimuli .
How might understanding CASP3 regulation impact neurodegenerative disease research?
CASP3 is implicated in neurodegenerative conditions, particularly Alzheimer's disease, where it cleaves amyloid-beta 4A precursor protein. The p53-miR-31-CASP3 regulatory axis presents several implications for neurodegenerative research:
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New targets for therapeutic intervention to prevent aberrant CASP3 activation in neurons
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Biomarkers for disease progression based on pathway components
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Understanding selective neuronal vulnerability through differences in regulatory mechanisms
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Potential for protective preconditioning strategies in at-risk individuals
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Development of models that better recapitulate disease processes by incorporating this regulatory axis
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Personalized approaches based on individual variations in this pathway
Further research into how this pathway functions specifically in neuronal contexts could yield significant insights into neurodegenerative disease mechanisms and potential interventions .
Product Science Overview
Caspases are classified into two main categories based on their roles: initiator caspases (e.g., caspase-8 and caspase-9) and executioner caspases (e.g., caspase-3, caspase-6, and caspase-7) . Caspase 3 is an executioner caspase, meaning it is responsible for the cleavage of various key cellular proteins, leading to the morphological and biochemical changes observed during apoptosis .
Caspase 3 exists as an inactive proenzyme (procaspase) that undergoes proteolytic processing at conserved aspartic residues to produce two subunits, large and small, which dimerize to form the active enzyme . The active site of caspase 3 contains a cysteine residue (Cys-163) and a histidine residue (His-121), which are crucial for its enzymatic activity .
Caspase 3 is involved in the cleavage and activation of other caspases, such as caspase-6 and caspase-7, and is itself activated by caspases-8, -9, and -10 . It plays a central role in the execution phase of apoptosis by cleaving various cellular substrates, including poly (ADP-ribose) polymerase (PARP), which is involved in DNA repair, and the amyloid-beta precursor protein, which is associated with neuronal death in Alzheimer’s disease .
The activation of caspase 3 can occur through two main apoptotic pathways: the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway . In the intrinsic pathway, cytochrome c released from mitochondria binds to Apaf-1 and procaspase-9 to form the apoptosome, leading to the activation of caspase-9, which in turn activates caspase 3 . In the extrinsic pathway, the binding of ligands to death receptors (e.g., Fas) leads to the formation of the death-inducing signaling complex (DISC), which activates caspase-8, subsequently activating caspase 3 .
Caspase 3 activity is tightly regulated by various mechanisms to ensure controlled apoptosis. Inhibitors of apoptosis proteins (IAPs) can bind to and inhibit active caspases, including caspase 3 . Additionally, the expression of caspase 3 can be regulated at the transcriptional level by various factors, including p53, a tumor suppressor protein that can induce apoptosis in response to DNA damage .
Human recombinant caspase 3 produced in Sf9 insect cells is used for research purposes to study its structure, function, and role in apoptosis. The recombinant enzyme retains the same biological properties and functions as the native enzyme, making it a valuable tool for investigating the mechanisms of apoptosis and for developing potential therapeutic interventions targeting caspase 3 .
