EPGN Human, His
Description
Functional Roles and Mechanisms
EPGN signals through the epidermal growth factor receptor (EGFR/ErbB1), albeit with 100-fold lower affinity compared to EGF . Key functions include:
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Mitogenic Activity: Stimulates fibroblast and epithelial cell proliferation via EGFR phosphorylation and MAPK activation .
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Tissue Expression: Highly expressed in testis, liver, heart, and cancer cells (e.g., breast, prostate) .
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Pathological Links: Overexpression correlates with tumor growth and sebaceous gland hyperplasia in transgenic mice .
Mechanistic Insights
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Receptor Binding: Despite low EGFR affinity, EPGN induces prolonged receptor activation due to inefficient ubiquitylation and endocytosis .
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Crosstalk with Cancer Pathways: Elevated EPGN and TGFA levels predict resistance to cetuximab (an anti-EGFR antibody) in head/neck cancers .
Key Studies
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Transgenic Mouse Models:
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Cancer Research:
Comparative Analysis of EGFR Ligands
| Ligand | Receptor Affinity | Mitogenic Potency | Key Tissues |
|---|---|---|---|
| EPGN | Low | High | Testis, liver, tumors |
| EGF | High | Moderate | Ubiquitous |
| Epiregulin | Moderate | High | Placenta, keratinocytes |
| Data synthesized from |
Clinical and Therapeutic Implications
Product Specs
To ensure long-term stability, adding a carrier protein (0.1% HSA or BSA) is advisable.
Repeated freezing and thawing should be avoided.
Q&A
How should recombinant EPGN Human with His-tag be stored to maintain stability?
Recombinant EPGN Human produced in Escherichia coli or Sf9 insect cells requires strict storage protocols to prevent degradation. Lyophilized EPGN from E. coli (7.9 kDa) should be stored desiccated at -20°C , whereas insect cell-derived EPGN (10.8 kDa) is stabilized in phosphate-buffered saline (pH 7.4) with 10% glycerol. For long-term storage (>4 weeks), freezing at -20°C is recommended, with the addition of a carrier protein (0.1% HSA or BSA) to prevent aggregation . Repeated freeze-thaw cycles must be avoided, as they reduce bioactivity by disrupting tertiary structure.
Table 1: Stability Conditions for EPGN Variants
| Expression System | Molecular Weight | Storage Buffer | Long-Term Stability |
|---|---|---|---|
| E. coli | 7.9 kDa | Lyophilized | -20°C (desiccated) |
| Sf9 Insect Cells | 10.8 kDa | PBS + 10% glycerol | -20°C with 0.1% BSA |
What experimental approaches validate EPGN’s mitogenic activity in epithelial cells?
EPGN’s bioactivity is typically confirmed through phosphorylation assays targeting ErbB1 and downstream MAP kinases. A standard protocol involves:
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Cell Stimulation: Treat serum-starved epithelial cells (e.g., HaCaT) with 10–100 ng/mL EPGN for 15–30 minutes .
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Western Blotting: Probe lysates with anti-phospho-ErbB1 (Tyr1068) and anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibodies.
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Dose-Response Analysis: Quantify signal intensity to establish EC₅₀ values, ensuring linearity within the dynamic range .
Discrepancies in activity between batches may arise from improper folding or glycosylation differences (e.g., E. coli-derived EPGN lacks glycosylation, unlike insect cell variants) .
How do expression systems (E. coli vs. insect cells) influence EPGN functionality?
The choice of expression system impacts post-translational modifications and receptor binding:
What strategies resolve contradictions in EPGN’s low ErbB1 affinity versus high mitogenic potency?
Despite EPGN’s low ErbB1 binding affinity , its mitogenic efficacy parallels high-affinity EGF ligands. This paradox is attributed to:
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Receptor Recycling Dynamics: EPGN induces inefficient ErbB1 ubiquitylation and endocytosis, prolonging receptor activation .
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Co-Receptor Synergy: Cross-talk with ErbB2/ErbB3 heterodimers amplifies signaling cascades.
To test these hypotheses:
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Perform receptor internalization assays using fluorescently labeled EPGN and time-lapse microscopy.
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Silence ErbB2/ErbB3 via siRNA and measure MAPK activation thresholds.
How can structural variations in His-tagged EPGN affect cross-linking studies?
The C-terminal His-tag (6xHis) in EPGN (e.g., Sf9-derived sequence: ADPAAVTVTP...KHHHHHH ) may sterically hinder interactions in cross-linking experiments. Mitigation strategies include:
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Tag Removal: Use thrombin or TEV protease to cleave the His-tag post-purification.
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Alternative Tagging: N-terminal tags or engineered linkers (e.g., GGGS repeats) minimize interference.
Validate tag removal via MALDI-TOF MS or anti-His Western blotting to confirm cleavage efficiency.
What advanced MS techniques characterize EPGN’s post-translational modifications (PTMs)?
Insect cell-derived EPGN contains N-linked glycosylation at Asn¹⁵ and Asn²⁷ , which modulate receptor binding. To map PTMs:
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Trypsin Digestion: Fragment EPGN and analyze peptides via LC-MS/MS.
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Glycan Profiling: Use PNGase F to release N-glycans, followed by hydrophilic interaction chromatography (HILIC)-MS.
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Data Analysis: Tools like Byonic or Glycomod identify glycan compositions and occupancy sites.
Table 2: Common PTMs in EPGN Variants
Product Science Overview
Epigen was identified through amino acid sequence homology with other EGF family members. The sequence was initially obtained from mouse keratinocyte mRNA . Human Epigen cDNA encodes a 154 amino acid residue type I transmembrane precursor glycoprotein. This glycoprotein consists of a 22 amino acid signal peptide, an 88 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 23 amino acid cytoplasmic domain .
Epigen is expressed in normal human mammary tissue and in certain prostate and breast carcinomas . It binds to known EGF receptors, albeit with lower affinity compared to EGF. Despite this, Epigen is superior in inducing cell proliferation . It induces responses in MDA-MB-468 breast carcinoma cells and has effects on prostate cells and vascular endothelial cells . Epigen binds to ErbB1/Her1, causing receptor homodimerization and heterodimerization with other Erb/Her receptors .
Recombinant human Epigen (hEpigen) is produced in E. coli. The recombinant protein is typically purified to a high degree of purity, often greater than 98% as determined by SDS-PAGE . The bioactivity of recombinant hEpigen is measured in cell proliferation assays, with an ED50 typically between 2-30 ng/mL .
Recombinant Epigen is used in various research applications, particularly in studies related to cell proliferation and cancer research. It is important to note that products containing recombinant Epigen are labeled for research use only and are not approved for diagnostic or therapeutic purposes .
Epigen’s role in cell proliferation and its potential implications in cancer research make it a significant protein for further study and understanding.
