EGF Long Human
Description
Production and Purification
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Expression: Produced recombinantly in E. coli, enabling scalable, serum-free manufacturing .
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Purification: Proprietary chromatographic techniques yield >95% purity .
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Formulation: Lyophilized from 10 mM HCl, reconstituted in 100 mM acetic acid for optimal solubility .
Biological Activity
EGF Long binds the ErbB1/EGFR receptor, activating downstream signaling pathways (e.g., MAPK, PI3K-AKT) to promote cell proliferation and differentiation . Key metrics:
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Synergy: Enhances growth when combined with insulin-like growth factors (e.g., LONG® R3 IGF-I) in HEK293, MDCK, and HeLa cells .
Applications in Research and Therapeutics
EGF Long is utilized in:
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Therapeutic Cell Culture: Supports serum-free expansion of fibroblasts and epithelial cells .
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Keratinocyte Studies: Drives differentiation and growth in skin models .
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Vaccine Production: Facilitates scalable manufacturing of cell-based vaccines .
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Cancer Research: Used to study EGFR signaling pathways without promoting malignant transformation in preclinical models .
Comparative Advantages Over Native EGF
Research Gaps and Future Directions
Product Specs
Q&A
What structural and functional distinctions exist between EGF Long Human and native EGF?
EGF Long Human incorporates a 53-residue N-terminal extension peptide that increases molecular mass to 12.3 kDa compared to native EGF's 6.2 kDa . This modification:
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Enhances protease resistance during prolonged cell culture
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Maintains receptor binding affinity (Kd ≈ 0.1-1 nM range) through conserved C-terminal domain preservation
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Requires acidic reconstitution buffers (100 mM acetic acid) to prevent aggregation
Comparative studies show equivalent phosphorylation of EGFR Tyr-1068 within 5 minutes post-stimulation, confirming conserved signaling initiation .
What are the optimal protocols for reconstituting and storing EGF Long Human?
Standardized handling protocols derived from stability studies :
| Parameter | Specification |
|---|---|
| Reconstitution | 100 mM acetic acid ≥100 µg/ml |
| Short-term storage | 4°C for ≤7 days in acidified solution |
| Long-term storage | -80°C lyophilized with 0.1% HSA carrier |
| Freeze-thaw cycles | ≤3 cycles with <15% activity loss |
Bioactivity validation should employ EGFR phosphorylation assays rather than mere mass spectrometry, as improper folding reduces signaling potency despite intact molecular weight .
How should researchers resolve contradictory mitogenic activity data across cell lines?
Discrepancies in proliferation assays (e.g., 3T3 fibroblasts vs. HaCaT keratinocytes) stem from:
Experimental Design Considerations
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Receptor density variations: Flow cytometry quantifies surface EGFR levels pre-stimulation
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Autocrine loop interference: CRISPR knockout of endogenous EGF in test cells
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Temporal response profiles: Pulse stimulation (15min) vs. continuous exposure
Methodological Optimization Table
| Challenge | Solution | Validation Metric |
|---|---|---|
| Proteolytic degradation | PEGylation at K48/K49 | LC-MS/MS peptide mapping |
| Thermal denaturation | Trehalose-based cryoprotectants | Circular dichroism at 222 nm |
| pH instability | Hepes-buffered saline (pH 7.4) | Dynamic light scattering |
Controlled release matrices (e.g., PLGA microspheres) maintain effective concentrations >1 ng/ml for 14 days in vivo .
Does the N-terminal extension alter receptor dimerization kinetics?
Surface plasmon resonance (SPR) analyses reveal:
Binding Parameters
| Construct | ka (1/Ms) | kd (1/s) | KD (nM) |
|---|---|---|---|
| Native EGF | 2.1×10⁶ | 8.3×10⁻⁴ | 0.40 |
| EGF Long | 1.8×10⁶ | 7.9×10⁻⁴ | 0.44 |
While equilibrium binding remains comparable, fluorescence correlation spectroscopy shows 2.3-fold slower lateral mobility in membrane-proximal EGFR complexes, suggesting steric effects from the extension .
What long-term exposure risks exist based on preclinical models?
28-day rodent studies demonstrate:
Key Findings from Toxicology Studies
| Parameter | EGF Long | Native EGF |
|---|---|---|
| Epithelial hyperplasia incidence | 92% | 88% |
| Dysplasia occurrence | 0% | 0% |
| Reversibility period | 14 days | 10 days |
Notably, co-administration with carcinogens (e.g., DMBA) showed no synergistic tumorigenesis except in hamster forestomach models (p=0.03) .
How do fusion tag systems improve EGF Long experimentation?
HaloTag fusion constructs enable:
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Single-step purification with >95% yield vs. traditional chromatography
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Covalent surface immobilization for controlled microenvironments
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Pulse-chase tracking via fluorescent ligands
Experimental Workflow
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Transform BL21(DE3) with pET-Halo-rhEGF
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Induce with 0.5 mM IPTG at 16°C
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Purify using HaloLink Resin
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Quantify activity via BRET-based EGFR internalization assay
This system reduces endotoxin contamination to <0.1 EU/µg versus 0.5 EU/µg in standard preps .
What controls are essential for EGF Long dose-response studies?
Required Control Matrix
| Control Type | Purpose | Frequency |
|---|---|---|
| Ligand-free | Baseline signaling | All experiments |
| TGF-α (10 ng/ml) | System competency | Per plate |
| Phosphatase inhibitor | Signal decay control | Every 2 hours |
| Temperature-matched vehicle | Solvent effects | Per condition |
For xCELLigence RTCA systems, normalize impedance values to 0-hour readings to account for differential adhesion effects .
Product Science Overview
LONG® EGF is produced in Escherichia coli (E. coli) and is free of animal-derived components . It is manufactured according to current Good Manufacturing Practices (cGMP) standards, ensuring high purity and consistency . The production facility is regularly audited by European and US contract manufacturers and biopharmaceutical companies .
The recommended concentration range for LONG® EGF in media is 10 - 50 μg/l, with a maximum concentration not exceeding 100 μg/l . It is advised to reconstitute the vial to the recommended concentration of 1 mg/ml in 10 mM HCl . Care should be taken to perform all activities in a controlled environment using aseptic techniques .
