PEDF Human, HEK

What is the molecular structure and key properties of human PEDF produced in HEK293 cells?

Human PEDF produced in HEK293 cells is a single, glycosylated polypeptide chain containing 410 amino acids with a molecular mass of approximately 45.6 kDa . The recombinant protein is typically fused to a tag (such as an 11 amino acid FLAG tag) at the C-terminus to facilitate purification and detection . Its amino acid sequence includes multiple functional domains responsible for its diverse biological activities.

The protein belongs to the serpin family but lacks serine protease inhibitory activity as it does not undergo the characteristic S (stressed) to R (relaxed) conformational transition typical of active serpins . This structural characteristic is important when considering its biological mechanisms of action in experimental designs.

Storage and handling considerations are critical for maintaining protein integrity. The lyophilized form is stable at room temperature for approximately 3 weeks and at -20°C for at least 2 years. Once reconstituted, it should be stored at 4°C for no more than 7 days or at -20°C for up to six months, with the addition of a carrier protein (0.1% HSA or BSA) recommended for long-term storage at -80°C .

How does recombinant PEDF function in cellular systems and what are its primary biological activities?

Recombinant PEDF exhibits multiple biological activities that can be studied in cellular systems:

• Anti-angiogenic activity: PEDF strongly suppresses angiogenesis by inhibiting the proliferation and differentiation of endothelial cells . In experimental systems, PEDF antagonizes pro-angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) . When designing angiogenesis assays, researchers should consider using both positive controls (VEGF) and PEDF to demonstrate the counteracting effects.

• Anti-tumorigenic effects: PEDF directly inhibits cancer cell proliferation in various tumor types including lung cancer, melanoma, pancreatic cancer, hepatoblastoma, prostate cancer, retinoblastoma, and colon cancer . In vitro studies have shown that PEDF can promote apoptotic cell death in melanoma cells and induce differentiation in neuroblastoma and prostate cancer cells .

• Neurotrophic activity: PEDF induces extensive neuronal differentiation, particularly in cells derived from the retina and central nervous system (CNS) . This activity makes it valuable for neurodevelopmental and neuroprotection studies.

• Anti-inflammatory properties: PEDF is involved in inflammatory responses, which should be considered when designing experiments in inflammation-related disease models .

When studying these functions, it is critical to use appropriate cellular models. For example, endothelial cell tube formation assays for anti-angiogenic studies, cancer cell line proliferation and apoptosis assays for anti-tumorigenic effects, and neuronal differentiation assays for neurotrophic studies.

What methodologies are recommended for reconstitution and application of lyophilized PEDF in cell culture experiments?

For optimal results in cell culture experiments with lyophilized PEDF, follow these methodological considerations:

• Reconstitution protocol: Add deionized water to the lyophilized protein to achieve a working concentration of 0.5 mg/ml and allow the pellet to dissolve completely . The reconstitution should be performed gently to avoid protein denaturation.

• Sterility considerations: Since commercial PEDF products are typically not sterile, filter the reconstituted protein using an appropriate sterile filter (0.22 μm) before using it in cell culture applications .

• Experimental concentrations: Based on research literature, effective PEDF concentrations in cell culture typically range from 10-100 ng/ml depending on the cell type and experimental endpoint. Preliminary dose-response experiments are recommended to determine optimal concentrations for your specific cellular model.

• Vehicle controls: Include proper controls in experiments, such as the same buffer without PEDF, to account for potential buffer effects on cellular responses.

• Timing considerations: PEDF's biological effects may be time-dependent. For anti-angiogenic studies, 24-72 hour treatment periods are commonly used. For neurotrophic effects, longer exposure (3-7 days) may be necessary to observe cellular differentiation.

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as they can compromise protein activity . Aliquot the reconstituted protein for single-use applications.

How does PEDF signaling differ across various cancer types and what methodological approaches best capture these differences?

PEDF signaling varies significantly across cancer types, requiring tailored methodological approaches:

• Receptor expression analysis: PEDF interacts with at least two known receptors: PEDF-R (ATGL/PNPLA2) for anti-angiogenic effects and PLXDC2, which is expressed in various cancer types including colon cancer, hepatocellular carcinoma, laryngeal cancer, testicular seminoma, and vulvar squamous cell carcinoma . Before studying PEDF effects, characterize receptor expression in your cancer model using RT-qPCR, western blotting, or immunohistochemistry.

• Cancer-specific signaling pathways: Different cancer types may exhibit varied responses to PEDF due to the activation of different downstream signaling cascades. For example:

  • In melanoma, PEDF promotes apoptosis through specific pathways

  • In neuroblastoma, it induces neuron-like differentiation

  • In prostate cancer, it promotes neuroendocrine differentiation

• Methodological considerations for cancer type differentiation:

  • Use paired PEDF-high and PEDF-low expressing cell lines from the same cancer type

  • Employ receptor-blocking antibodies to distinguish receptor-specific effects

  • Analyze downstream signaling using phosphorylation arrays or targeted western blots for key pathway markers

  • Consider 3D culture systems to better recapitulate tumor microenvironment interactions with PEDF

What are the optimal protocols for studying PEDF's dual role in anti-angiogenesis and direct anti-tumor activity in complex experimental models?

To effectively study PEDF's dual anti-angiogenic and anti-tumor roles, consider these advanced methodological approaches:

• Co-culture systems: Establish co-cultures of cancer cells with endothelial cells to simultaneously assess both direct anti-tumor effects and anti-angiogenic activities.

  • Method: Seed cancer cells in the bottom chamber of a transwell system and endothelial cells in the upper chamber

  • Treatment: Apply PEDF at 50-100 ng/ml to both compartments

  • Analysis: Measure cancer cell proliferation/apoptosis and endothelial tube formation concurrently

• 3D tumor spheroid models with embedded endothelial cells:

  • Generate tumor spheroids incorporating both cancer cells and endothelial cells (labeled with different fluorescent markers)

  • Treat with PEDF and analyze:

    • Changes in spheroid size and integrity (tumor effect)

    • Endothelial network formation within spheroids (angiogenesis effect)

    • Cell type-specific apoptosis markers

• In vivo models with dual readouts:

  • Xenograft models with complementary analyses:

    • Tumor volume measurements (direct anti-tumor effect)

    • Immunohistochemistry for CD31+ blood vessel density (anti-angiogenic effect)

    • Correlate these parameters with local PEDF concentrations

• Mechanistic separation studies:

  • Use PEDF-derived peptides that selectively target either anti-angiogenic or anti-tumor activities

  • Compare full-length PEDF with peptide fragments to delineate the contribution of each activity

  • Employ receptor-specific blocking antibodies to distinguish signaling pathways

• Signaling pathway analysis:

  • In endothelial cells, focus on VEGF-R pathway inhibition, Fas-FasL-mediated apoptosis

  • In tumor cells, examine pathways related to proliferation inhibition and apoptosis induction

  • Use pharmacological inhibitors of key pathway components to verify mechanisms

These approaches allow for comprehensive analysis of PEDF's multifaceted activities in complex cancer models, providing more physiologically relevant insights than simple monoculture systems.

How can researchers effectively investigate potential synergistic effects between PEDF and conventional cancer therapies?

Investigating PEDF's synergistic potential with conventional cancer therapies requires systematic methodological approaches:

• Combination treatment design:

  • Use dose-response matrices (checkerboard assays) with varying concentrations of PEDF (10-200 ng/ml) and conventional therapies

  • Calculate combination indices using Chou-Talalay method to quantitatively determine synergism, additivity, or antagonism

  • Include proper single-agent controls and vehicle controls

• Temporal sequence optimization:

  • Test different treatment sequences:

    • PEDF pretreatment followed by conventional therapy

    • Simultaneous administration

    • Conventional therapy followed by PEDF

  • Monitor both immediate and delayed effects (24h, 48h, 72h, 7d)

• Mechanism-based combinations:

  • Pair PEDF with therapies based on complementary mechanisms:

    • Anti-angiogenic agents (bevacizumab): Assess enhanced vascular normalization

    • Cytotoxic agents (cisplatin, doxorubicin): Examine enhanced apoptosis

    • Targeted therapies (TKIs): Investigate pathway convergence

  • Use pathway inhibitors to identify synergistic nodes

• Resistance model testing:

  • Develop resistant cell lines to conventional therapies

  • Determine if PEDF can resensitize resistant cells

  • Identify biomarkers that predict synergistic responses

• In vivo validation approaches:

  • Use xenograft or syngeneic tumor models

  • Compare:

    • Tumor growth kinetics

    • Survival outcomes

    • Tumor vasculature changes

    • Immune infiltration

• Translational biomarker development:

  • Identify expression signatures that predict combination efficacy

  • Develop assays for PEDF receptor status as potential companion diagnostics

This systematic approach allows researchers to comprehensively characterize potential synergistic effects and develop rational combination strategies for clinical translation.

What are the key physical and biochemical properties of recombinant human PEDF expressed in HEK293 cells?

The following table summarizes the key physical and biochemical properties of recombinant human PEDF expressed in HEK293 cells, essential for experimental planning and troubleshooting:

Understanding these properties is essential for appropriate experimental design, accurate interpretation of results, and troubleshooting potential issues in PEDF-related research.

How do different storage and handling conditions affect PEDF functionality in experimental systems?

Storage and handling conditions significantly impact PEDF functionality, requiring careful consideration in experimental design:

• Temperature effects on stability:

  • Room temperature exposure should be limited to less than 3 weeks for lyophilized protein

  • Reconstituted protein rapidly loses activity at room temperature and should be kept on ice during experiments

  • Long-term storage at -80°C with the addition of carrier proteins (0.1% HSA or BSA) provides optimal stability

• Freeze-thaw cycle impact:

  • Each freeze-thaw cycle can reduce biological activity by 10-30%

  • Activity loss manifests as reduced potency in functional assays rather than changes observable by SDS-PAGE

  • Single-use aliquots prevent activity loss from repeated freeze-thaw cycles

• Buffer composition considerations:

  • PEDF activity is optimal in physiological pH range (7.2-7.6)

  • High salt concentrations (>150mM) may reduce protein-protein interactions

  • Divalent cations (Ca²⁺, Mg²⁺) at 1-2mM can enhance stability

  • Reducing agents should be avoided as they may disrupt structural disulfide bonds

• Carrier protein addition effects:

  • Addition of 0.1% HSA or BSA prevents adhesion to storage vessels and enhances stability

  • Carrier proteins must be accounted for in total protein quantification assays

  • For some sensitive applications, carrier-free preparations may be necessary

• Container material considerations:

  • Low protein binding materials (polypropylene) are preferred over glass or polystyrene

  • Siliconized tubes minimize protein adsorption to container walls

  • Protein concentration should be verified after transfer between containers

Implementing these handling practices ensures maximum PEDF activity in experimental systems and enhances reproducibility across studies.

What are the most effective experimental models for studying PEDF's role in different pathological conditions?

Different pathological contexts require specialized experimental models to effectively study PEDF's role:

• Cancer research models:

  • Cell-based models: Compare PEDF effects across panels of cancer cell lines from different origins to identify cancer-specific responses

  • 3D organoid models: Patient-derived organoids better recapitulate tumor heterogeneity and microenvironment

  • In vivo models: Orthotopic xenografts provide physiologically relevant tumor microenvironments

  • Genetic models: PEDF knockout or overexpression in transgenic animals reveals context-dependent functions

• Ocular disease models:

  • Retinal explant cultures: Maintain retinal architecture while allowing controlled PEDF administration

  • Laser-induced choroidal neovascularization: Models age-related macular degeneration (AMD)

  • Streptozotocin-induced diabetic retinopathy: Allows study of PEDF in diabetic complications

  • Oxygen-induced retinopathy: Useful for studying retinopathy of prematurity

• Neurodegenerative disease models:

  • Primary neuronal cultures: Assess direct neuroprotective effects

  • Organotypic brain slice cultures: Preserve neural circuitry while allowing experimental manipulation

  • MPTP-induced Parkinson's model: Study PEDF's neurotrophic properties in dopaminergic neurons

  • Glutamate excitotoxicity models: Examine neuroprotective mechanisms

• Cardiovascular disease models:

  • Endothelial cell-smooth muscle cell co-cultures: Study vascular homeostasis

  • Ex vivo aortic ring assays: Quantify angiogenic sprouting

  • Ischemia-reperfusion injury models: Assess cardioprotective effects

  • Atherosclerosis models: Examine PEDF's role in plaque development

For each model system, appropriate controls are essential:

• PEDF neutralizing antibodies to confirm specificity

• Receptor antagonists to verify signaling mechanisms

• Comparison with established drugs in the therapeutic area

The selection of experimental models should be guided by the specific pathological process under investigation and the particular aspect of PEDF biology being studied.

How can researchers accurately quantify and validate PEDF expression and activity in experimental samples?

Accurate quantification and validation of PEDF expression and activity require multi-modal approaches:

• Protein expression quantification:

  • ELISA: Commercial kits offer sensitivity down to 20-50 pg/ml for quantifying PEDF in supernatants, plasma, or tissue lysates

  • Western blotting: Use monoclonal antibodies against specific PEDF epitopes; include recombinant PEDF standards for semi-quantitative analysis

  • Immunohistochemistry/Immunofluorescence: For spatial localization in tissues; validate antibody specificity with PEDF-knockout controls

  • Mass spectrometry: For absolute quantification and identification of post-translational modifications

• mRNA expression analysis:

  • RT-qPCR: Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • RNA-seq: For global expression analysis in the context of related pathways

  • In situ hybridization: For spatial mRNA localization in intact tissues

• Functional activity assays:

  • Endothelial cell tube formation inhibition: Quantify reduction in tube length/branching on Matrigel

  • Migration assays: Measure inhibition of endothelial or tumor cell migration

  • Apoptosis assays: Quantify caspase activation or Annexin V staining in target cells

  • Neurite outgrowth assays: Measure neurite length in neuronal cultures

• Receptor binding and signaling validation:

  • Surface plasmon resonance: Determine binding kinetics to PEDF receptors

  • Receptor phosphorylation: Assess PEDF-induced signaling through western blotting

  • Reporter gene assays: Measure transcriptional responses to PEDF treatment

• Validation strategies:

  • Genetic approaches: Use CRISPR/Cas9 to knockout/knockdown PEDF or its receptors

  • Pharmacological approaches: Apply receptor antagonists or downstream pathway inhibitors

  • Neutralizing antibodies: Block PEDF activity to confirm specificity of observed effects

  • Structure-function studies: Use PEDF fragments to map functional domains

These complementary approaches provide comprehensive assessment of PEDF expression and activity, enhancing confidence in experimental findings and facilitating mechanistic insights.

What are common experimental pitfalls when working with PEDF and how can researchers address them?

Researchers working with PEDF frequently encounter several experimental challenges that can be addressed with specific strategies:

• Inconsistent biological activity:

  • Problem: Variable responses between experiments despite using the same PEDF concentration

  • Solutions:

    • Validate each new lot with a standardized functional assay

    • Include positive controls (e.g., anti-VEGF antibody for anti-angiogenic assays)

    • Standardize cell passage numbers and culture conditions

    • Monitor storage conditions and avoid freeze-thaw cycles

• Protein adsorption to surfaces:

  • Problem: Loss of active PEDF due to adherence to plasticware

  • Solutions:

    • Add carrier protein (0.1% BSA) to dilution buffers

    • Use low-protein binding tubes and plates

    • Pre-coat surfaces with BSA solution

    • Verify final concentrations using ELISA after dilution

• Discrepancies between different detection methods:

  • Problem: Different antibodies yield inconsistent results in western blot vs. ELISA

  • Solutions:

    • Verify antibody specificity with recombinant protein controls

    • Use antibodies recognizing different epitopes to confirm results

    • Consider post-translational modifications affecting epitope recognition

    • Include appropriate negative controls (PEDF knockout/knockdown)

• Interference from endogenous PEDF:

  • Problem: Baseline PEDF production by experimental cells masks exogenous PEDF effects

  • Solutions:

    • Measure endogenous PEDF production in your model system

    • Use CRISPR/Cas9 to generate PEDF-knockout cell lines

    • Use tagged recombinant PEDF to distinguish from endogenous protein

    • Employ serum-free conditions when possible to eliminate serum-derived PEDF

• Species-specific differences:

  • Problem: Human PEDF shows variable activity across species

  • Solutions:

    • Use species-matched PEDF when possible

    • Validate cross-species activity with functional assays

    • Consider receptor expression and conservation across species

    • Include appropriate species-specific positive controls

• Cellular context variability:

  • Problem: PEDF effects vary dramatically between cell types

  • Solutions:

    • Verify receptor expression in each cell system

    • Conduct dose-response curves for each new cell type

    • Consider cell density effects on PEDF responsiveness

    • Account for receptor desensitization in prolonged experiments