Recombinant Human Pro-epidermal growth factor (EGF), partial (Active)
Description
Biological Activity
Functional Roles:
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Cell Proliferation: Induces mitogenesis in epithelial cells, fibroblasts (ED₅₀ <1 ng/mL in Balb/c 3T3 cells)
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Magnesium Regulation: Activates TRPM6 channels in renal tubules
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Wound Healing: Accelerates fibroblast migration (50 ng/mL reduces scratch gap by >60% in 48 hr)
Mechanism:
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Binds EGFR/ErbB1, triggering receptor dimerization and tyrosine kinase signaling
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Promotes disulfide-dependent structural stability (3 conserved bonds)
Research Applications
In Vitro Models:
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Fibroblast Migration: 48 hr scratch closure rates:
Growth Factor Closure Rate (%) EGF 85 Epiregulin 78 Betacellulin 65
Therapeutic Studies:
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Diabetic Ulcers: 76% healing rate with 0.005% topical EGF (n=68)
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Anti-Inflammatory Effects: Suppresses M1 macrophage-induced fibroblast proliferation at 10 ng/mL
Production & Quality Control
Expression Challenges:
Analytical Validation:
Product Specs
Target Background
- Our findings demonstrate the exceptional efficacy of the chimeric EGFETA toxin against EGFR-positive cancers, highlighting its potential for further development as a targeted therapy for EGFR-positive tumors resistant to monoclonal antibodies. PMID: 30226622
- This study underscores the potential role of EGF in promoting hepatocellular carcinoma (HCC) metastasis, elucidates a novel pathway regulating fibronectin expression, and identifies potential targets for HCC prevention and treatment. PMID: 29315755
- The abnormally elevated expression of EGF and TGF-alpha is strongly associated with the occurrence and progression of chronic pancreatitis and pancreatic cancer. PMID: 29125273
- ERRa positively regulates the cell proliferation, migration, and invasion of colon cancer cells. Notably, suppression of ERRa completely abolishes the EGF treatment-induced proliferation of colon cancer cells. PMID: 30185207
- EGF significantly upregulates RFPL3 and hTERT protein levels in non-small cell lung cancer cells. Pretreatment with AG1478 and erlotinib effectively attenuates this upregulation induced by EGF. EGF promotes proliferation and inhibits apoptosis, while PD98059 decreases RFPL3 and hTERT protein expression. Overexpression of RFPL3 increases the expression of hTERT and related MEK pathway proteins. PMID: 29749533
- We have identified the novel N-72, which plays a crucial role in EGF-induced migration by targeting MMP2 in Human amnion mesenchymal stem cells (hAMSCs). PMID: 29734654
- Our findings suggest that the spleen regulates the functions of hematopoietic stem cells in cirrhotic hypersplenism by modulating EGF signaling. PMID: 29721775
- Upon blocking HIP1 expression using siRNAs, EGFR endocytosis is accelerated, an effect dependent on the EGF concentration. This endocytosis colocalizes with clathrin expression. Our results suggest that inhibiting HIP1 can accelerate the endocytosis and degradation of EGFR. PMID: 29039605
- This study demonstrates that EGF induces aggressiveness of gastric cancer cells by activating epithelial-to-mesenchymal transition, involving activation of the ERK1/2 pathway and subsequent uPAR expression. PMID: 28849196
- Our findings indicate that the EGF system acts as a mechanosensitizer in bone marrow stromal cells. PMID: 28843157
- EGF counteracts Tat modulation of human endogenous retroviruses of the W family in astrocytes. PMID: 28474333
- FTIR spectra analysis of EGF at various stages of synthesis, including unconjugated, post-treatment with alpha-lipoic acid, attached to gold nanoparticles, and bound to the bifunctional nanoprobe, revealed decreasing disordered structures and turns, and increasing loops as the process progressed. Notably, the final product exhibited an overall increase in beta-sheets compared to pure EGF, although this increase was not linear and fluctuated. PMID: 29122663
- EGF-mediated lysosome trafficking, protease secretion, and invasion are regulated by the activity of p38 mitogen-activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a mechanism distinct from that previously reported for HGF, suggesting the presence of redundant signaling pathways controlling lysosome positioning. PMID: 28978320
- While the diabetic chronic wounds microenvironment is hostile to local growth factor bioavailability, local EGF infiltration overcomes the limitations of topical application, expanding its therapeutic potential. Our clinical pharmacovigilance and basic studies validate the significance of local growth factor infiltration for chronic wound healing. PMID: 28904952
- This study provides the first evidence linking the EGF rs2298999 C/T polymorphism to gout. PMID: 27506295
- The increased EGFR expression observed in patients with seborrheic keratomas (SK) and concomitant type 2 diabetes mellitus (DM2) is attributed to insulin resistance and hyperinsulinemia. In this context, dysregulation of insulin signal transmission into the cell leads to alterations in EGF synthesis and signaling pathways that govern cell proliferation and growth. PMID: 28791994
- This research identifies a novel EGFR-NF-kappaB-FOXC1 signaling axis crucial for breast cancer cell function. PMID: 28629477
- EGFR pathway gene expression analysis reveals that DeltaNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Furthermore, the addition of EGF or neutralizing EGFR antibodies demonstrates that EGFR activation is responsible for DeltaNp63-mediated loss of cellular adhesion. PMID: 28349272
- EGF upregulates CCL2 expression in HNSCC cells, recruiting monocytes and transforming them into M2-like macrophages, thus forming a positive feedback paracrine loop. PMID: 27888616
- This study demonstrates that EGF induces epithelial-mesenchymal transition through phospho-Smad2/3-Snail signaling pathways in breast cancer cells. PMID: 27829223
- EGF and TNFalpha cooperatively promote the motility of HCC cells primarily through NF-kappaB/p65-mediated synergistic induction of fibronectin in vitro. These findings highlight the crosstalk between EGF and TNFalpha in promoting HCC and provide potential targets for HCC prevention and treatment. PMID: 28844984
- Data suggest that EGF induces colorectal cancer cells to undergo epithelial-mesenchymal transition, enhances their invasive/migratory capabilities, and promotes phosphorylation of Ezrin at Tyr353. PMID: 28535417
- Simulation results indicate that human epidermal growth factor receptor (hEGFR) soluble extracellular domains (sECD):EGF exhibit different dynamic properties at distinct pH values. The complex may have a higher tendency for activation at pH 8.5. PMID: 27179806
- EGF and IP-10 levels are significantly elevated, while GRO levels are lower in the tear profile of HIV patients with dry eye disease (DED) compared to immunocompetent patients with DED. PMID: 27585367
- Data, including those from studies using transgenic/knockout mice, suggest that surfactant protein A1 (SPA1) interferes with EGF binding to EGFR in pulmonary alveoli cell lines. SPA1 directly binds to the extracellular domain of EGFR, and this binding appears to differ from that of SPD to EGFR. Importantly, SPA1 binding to EGFR does not suppress EGF-induced phosphorylation of EGFR or cell proliferation. PMID: 28972165
- The interplay between EGF and AREG in airway basal cell stem/progenitor cells is a key mechanism mediating the interconnected pathogenesis of all major smoking-induced lesions in the human airway epithelium. PMID: 27709733
- Caspase-3 inhibitors suppress the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicate that EGF-F9 activates signals for apoptosis and induces de-adhesion in a caspase-3-dependent manner. PMID: 27129300
- Our findings provide evidence that CDK1/2 participate in regulating constitutive pre-mRNA splicing by EGF stimulation in MDA-MB-468 cells. PMID: 27109354
- The EGF rs4444903 GG genotype is associated with a higher susceptibility to HCV-related liver cirrhosis and hepatocellular carcinoma in the Chinese Han population. PMID: 28397482
- TGF-beta opposes EGF-mediated sensitization to TRAIL-induced caspase-8 activation and apoptosis in non-transformed breast epithelial cells. EGF and TGF-beta delicately regulate the sensitivity of human breast epithelial cells to TRAIL, potentially playing a role during morphogenesis. PMID: 27208428
- Amplification of the EGFR gene can be maintained and modulated by variations in EGF concentrations in in vitro models of glioblastoma multiforme. PMID: 28934307
- Our study showed that the EGF61 rs4444903GA genotype was associated with a decreased risk of non-syndromic cleft lip with or without cleft palate. Our findings provide further evidence regarding the role of EGF61 variations in the development of non-syndromic cleft lip with or without cleft palate in families of the studied populations. PMID: 28906376
- Interestingly, EGF rapidly downregulates LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT) expression by enhancing histone deacetylation at the respective promoter. PMID: 27485121
- EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2, and the PEST domain is critical for EGF/calpain actions. PMID: 28640887
- Salivary levels of EGF are significantly increased during the acute phase of natural rotavirus infection. PMID: 28558652
- Our findings have identified a role for members of these signaling pathways in regulating EGF-induced vimentin expression in the MDA-MB-468 breast cancer cell line. PMID: 27163529
- miR-223 downregulates the local expression of epidermal growth factor (EGF), leading to decreased activation of the EGF receptor (EGFR) on target cells. This ultimately dampens a positive EGF-EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. PMID: 26876200
- EGFR and EGF expression did not show a significant difference between placentas from normal pregnancies and those complicated with preeclampsia. PMID: 27657362
- Atomistic molecular dynamics simulations indicate that N-glycosylation of the EGFR extracellular domain plays critical roles in the binding of growth factors, monoclonal antibodies, and dimeric partners to the monomeric EGFR extracellular domain. PMID: 28486782
- CMTM3 decreases EGFR expression, facilitates EGFR degradation, and inhibits the EGF-mediated tumorigenicity of gastric cancer cells by enhancing Rab5 activity. PMID: 27867015
- Our findings suggest that EGF not only promotes the proliferation of adipose stem cells and delays their senescence but also maintains their differentiation potency. These effects are linked to EGF-induced activation of the STAT signaling pathway. PMID: 28746211
- The results demonstrate that the interaction between STS-1 and ShcA is regulated in response to EGF receptor activation. PMID: 28690151
- Insulin treatment leads to sustained Akt activity, while EGF or PDGF-AA promotes transient signaling. PDGF-BB produces sustained responses at higher concentrations. Transient responses to EGF are attributed to negative feedback at the receptor level, as a second treatment yields minimal responses. In contrast, parallel exposure to IGF-I results in full Akt activation. PMID: 27044757
- Our results indicate that different concentrations of bFGF and EGF supplemented during the propagation of neural rosettes influence the identity of the resulting neural cells. PMID: 27321088
- F25P preproinsulin effectively reduced blood concentrations of EGF, VEGF, and MMP-9 in tumor-bearing mice with EGFR-mutant glioblastoma. PMID: 27317648
- The conformational stability of the EGFR as influenced by glycosylation, dimerization, and EGF hormone binding has been described. PMID: 28019699
- Differential expression patterns of EGF, EGFR, and ERBB4 are essential for epithelial restitution and remodeling in nasal epithelium. PMID: 27285994
- Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate that the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform. PMID: 27314851
- Data suggest that activated platelets release ADAMDEC1, which hydrolyzes pro-EGF (epidermal growth factor) to soluble, active HMW-EGF. Proteolytic cleavage of pro-EGF initially occurs at the C-terminal arginyl residue of the EGF domain. This proteolysis is the regulated, rate-limiting step in generating soluble EGF from activated platelets. PMID: 28455445
- Subgroup analysis in a Slovak population by gender showed that the EGF G61G genotype and allele G were associated with a non-significantly increased risk of major depressive disorder (MDD). PMID: 27755861
Q&A
What is the structural organization of recombinant human EGF and how does it affect production strategies?
Recombinant human EGF contains an EGF domain with the characteristic sequence CX7CX4-5CX10-13CXCX8C, where cysteines form three disulfide bonds in the combinations C1-C3, C2-C4, and C5-C6. This structural feature presents significant challenges for recombinant production in prokaryotic systems, as improper disulfide bond formation often leads to misfolding and inclusion body formation .
Successful production strategies must account for proper disulfide bond formation to ensure biological activity. Mass spectrometry analyses have confirmed correct disulfide bonds for several EGF family members, including AREG (all three bonds), BTC (C1-C2 pairs), and EPGN and EPR (C5-C6) . The complex structure necessitates careful quality control during production to verify proper folding.
How can researchers verify the correct folding and stability of recombinantly produced EGF?
Multiple analytical approaches should be employed to confirm proper folding of recombinant EGF:
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Limited proteolysis assays: Correctly folded EGF shows resistance to trypsin digestion when disulfide bonds are intact. In controlled experiments, reduced samples (treated with DTT) show complete degradation after 30 minutes of trypsin exposure, while non-reduced samples demonstrate significantly higher resistance. The appearance of intermediate bands in non-reduced samples typically indicates the presence of the EGF domain nucleus maintained by disulfide bonds .
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Circular dichroism (CD) spectroscopy: Properly folded EGF family members display characteristic CD spectra consistent with proteins containing low alpha-helical content .
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Thermal stability analysis: Techniques such as nanoDSF can assess conformational stability under thermal stress. Properly folded hEGF, hEPGN, hEPR, and hTGFα typically demonstrate strong resistance to aggregation at high temperatures .
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Functional assays: Cell proliferation and migration assays provide the ultimate verification of proper folding, as only correctly folded EGF maintains biological activity.
What expression systems are optimal for producing biologically active recombinant human EGF?
Different expression systems offer distinct advantages for recombinant human EGF production:
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E. coli expression system: Despite challenges with disulfide bond formation, E. coli remains a widely used system for EGF production due to its simplicity and cost-effectiveness. When optimized with appropriate fusion partners and culture conditions, it can yield biologically active EGF with correct disulfide bonding .
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HEK293 expression system: Mammalian cell expression in HEK293 cells produces highly active EGF with proper post-translational modifications. This system is particularly valuable for producing the pro-form of EGF with C-terminal tags (such as Fc-His) .
The choice of expression system depends on specific research requirements, including the need for post-translational modifications, tag presence, and required activity levels. For studies requiring precise activity measurements, HEK293-expressed EGF has demonstrated biological activity at concentrations as low as 0.065-0.26 ng/ml in fibroblast proliferation assays .
What purification strategies ensure high purity and biological activity of recombinant human EGF?
Successful purification of recombinant human EGF requires a multi-step approach:
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Initial capture: Affinity chromatography using tag-based approaches (His-tag, GST-tag) provides efficient initial purification.
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Tag removal: When fusion tags are used for expression, site-specific proteases (such as TEV or thrombin) can be employed for tag removal.
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Polishing steps: Size exclusion chromatography and/or ion exchange chromatography are essential for achieving high purity and removing aggregates.
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Quality control: SDS-PAGE (under reducing and non-reducing conditions), Western blotting, and mass spectrometry are crucial for verifying purity and integrity.
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Activity verification: Biological activity must be confirmed through cell-based assays measuring proliferation or receptor activation, as structural integrity does not always guarantee functional activity .
Researchers should note that while high purity is essential, the biochemical procedures should maintain the native conformation with intact disulfide bonds to preserve biological activity.
How can researchers accurately quantify the biological activity of recombinant human EGF?
Standardized assays for quantifying EGF biological activity include:
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Cell proliferation assays: The MTT method using HeLa cells or BALB/c 3T3 mouse embryonic fibroblasts provides reliable activity measurements. Active recombinant EGF typically shows a dose-dependent response, with effective concentrations ranging from 0.05-50 ng/ml .
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Receptor autophosphorylation: Measuring EGF Receptor autophosphorylation in A431 cells, with active EGF effectively enhancing receptor phosphorylation at concentrations of 1-10 ng/ml .
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Scratch wound healing assays: Using fibroblast cell lines (such as NDFH), artificial wounds are created in cell monolayers, and the rate of closure after EGF treatment is quantified. This assay measures combined proliferation and migration effects .
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Dose-response curves: Serial dilutions starting from 50 ng/ml can establish ED50 values, which for highly active recombinant EGF typically range from 0.065-0.26 ng/ml in fibroblast proliferation assays .
The following table shows representative proliferation data for EGF family members:
| EGF Family Member | Effective Concentration Range | Relative Potency in Proliferation Assays |
|---|---|---|
| hEPR | 0.5-50 ng/ml | Highest induction capacity |
| hBTC | 5-50 ng/ml | Very high induction |
| hAREG | 5-50 ng/ml | High induction |
| hEGF | 5-50 ng/ml | High induction |
| hTGFα | 0.05-50 ng/ml | Moderate to high induction |
| hEPGN | 0.05-50 ng/ml | Moderate induction |
| hHBEGF | 5-50 ng/ml | Lowest induction among family members |
Data adapted from functional analysis studies
What delivery systems can enhance the bioavailability of recombinant human EGF for research applications?
Several delivery systems have been developed to overcome the limited bioavailability of EGF due to its large molecular size (6045 Da):
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Transfersomal systems: Lipid vesicle formulations incorporating phospholipids and surfactants can significantly enhance skin penetration. Optimal formulations contain specific ratios between lipid vesicles and rhEGF (ranging from 100:1 to 200:1) .
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Emulgel preparations: Transfersome-containing emulgels have demonstrated enhanced penetration compared to non-transfersomal formulations, with increased penetration correlating with higher lipid content .
The table below shows key characteristics of rhEGF-loaded transfersomes:
| Formulation | Particle Size (nm) | Polydispersity Index | Zeta Potential (mV) | Entrapment Efficiency (%) |
|---|---|---|---|---|
| TF-EGF1 (200:1) | 128.1 ± 0.66 | 0.109 ± 0.004 | -43.1 ± 1.07 | 97.77 ± 0.09 |
| TF-EGF2 (133:1) | 125.4 ± 0.61 | 0.110 ± 0.008 | -36.8 ± 2.08 | 92.78 ± 2.11 |
| TF-EGF3 (100:1) | 118.7 ± 1.11 | 0.116 ± 0.007 | -40.5 ± 0.90 | 92.15 ± 0.38 |
Data presented as mean ± standard deviation (n = 3)
When designing delivery systems, researchers should consider the impact on EGF stability, release kinetics, and maintenance of biological activity.
What methodologies are used to evaluate recombinant human EGF efficacy in clinical research models?
Clinical evaluation of recombinant human EGF requires rigorous methodological approaches:
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Randomized controlled trial design: Double-blind, placebo-controlled studies provide the highest quality evidence. For example, in evaluating rhEGF for radiation-induced oral mucositis, a multi-institutional phase 2 trial randomized patients to receive placebo or one of three EGF doses (10, 50, or 100 μg/mL) .
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Standardized scoring systems: Using validated scales like the Radiation Therapy Oncology Group (RTOG) scoring criteria allows for objective assessment of outcomes .
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Defined response criteria: Clear definitions of treatment response (e.g., achieving RTOG grade ≤2 at specific timepoints) ensure consistency in data interpretation .
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Statistical analysis: Comparing response rates between treatment and control groups with appropriate statistical tests (e.g., demonstrating a 64% response with 50 μg/mL EGF versus 37% in control groups, p=0.0246) .
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Dose-response assessment: Testing multiple concentration levels (10, 50, and 100 μg/mL) to determine optimal dosing regimens .
These methodological approaches allow for robust evaluation of rhEGF efficacy while minimizing bias and confounding factors.
How can researchers distinguish between the effects of various EGF family members in experimental systems?
Distinguishing between effects of different EGF family members requires careful experimental design:
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Receptor specificity analysis: All seven EGF family members (hEGF, hEPR, hAREG, hBTC, hTGFα, hEPGN, hHBEGF) bind to ErbB1, but with different affinities and activation profiles. Receptor binding assays and phosphorylation studies can differentiate their interactions .
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Functional comparison: Comparative functional assays reveal distinct potencies. For example, in scratch healing assays, hEGF typically shows the highest rate of wound closure, followed by hEPR, hEPGN, hAREG, hBTC, hTGFα, and hHBEGF with the lowest closure percentage .
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Dose-response profiling: Some family members (hEPGN, hEPR, hTGFα) induce proliferation at concentrations as low as 0.5 ng/mL, while others (hEPGN, hTGFα) remain active at 0.05 ng/mL .
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Structural stability analysis: Resistance to proteolysis differs among family members, with hEGF, hEPR, and hTGFα showing the highest resistance, followed by hAREG and hEPGN, while hBTC and hHBEGF demonstrate greater sensitivity .
Understanding these distinctive characteristics allows researchers to select the appropriate EGF family member for specific experimental models and therapeutic applications.
How do disulfide bond patterns influence the structural stability and biological activity of recombinant human EGF?
The three disulfide bonds (C1-C3, C2-C4, C5-C6) in the EGF domain are critical determinants of both structural stability and biological activity:
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Structural integrity: Limited proteolysis experiments demonstrate that intact disulfide bonds provide significant resistance to enzymatic degradation. When these bonds are disrupted by reducing agents like DTT, EGF becomes highly susceptible to trypsin digestion, being completely degraded within 30 minutes .
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Conformational stability: Different EGF family members show varying stability patterns based on their disulfide bond arrangements. For example, hEGF, hEPGN, hEPR, and hTGFα demonstrate strong resistance to thermal denaturation, while hBTC, hAREG, and hHBEGF show intermediate resistance .
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Functional domains: Partial proteolysis of non-reduced samples reveals intermediate bands representing the EGF domain nucleus maintained by disulfide bonds. This structurally preserved core is likely responsible for receptor interaction and biological activity .
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Correlation with activity: The proper disulfide bond formation is essential for biological activity, as demonstrated by the high potency of correctly folded recombinant factors in proliferation assays at concentrations as low as 0.05-5 ng/ml .
Researchers investigating EGF structure-function relationships should employ methods like mass spectrometry to verify correct disulfide bond formation, particularly focusing on the C1-C3, C2-C4, and C5-C6 pairings.
What are the key methodological considerations when designing experiments to evaluate age-related changes in EGF signaling?
When investigating age-related changes in EGF signaling, researchers should consider several methodological aspects:
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Age standardization: Properly define "aged" experimental models. In murine studies, mice 18-22 months old are typically considered aged, while 6-8 week old mice serve as young controls .
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EGF level measurement: Quantify both circulating (serum) EGF levels and tissue-specific expression. Notably, studies have shown that serum EGF levels may not differ significantly between aged and young mice, suggesting that receptor function rather than ligand availability may be the primary determinant of age-related changes .
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Receptor functionality assessment: Evaluate EGFR expression, phosphorylation status, and downstream signaling pathway activation in response to standardized EGF stimulation.
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Tissue-specific responses: Consider that EGF responsiveness may vary significantly between tissue types in the aging process. For example, bone marrow hematopoietic stem cells (HSCs) from aged mice show distinct responses to EGF stimulation compared to young controls, with EGF capable of suppressing age-related myeloid skewing .
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Interaction with other signaling pathways: Assess cross-talk between EGF signaling and other pathways known to be altered in aging, including inflammatory mediators and oxidative stress responses.
These methodological considerations ensure robust experimental design when evaluating the complex relationship between aging and EGF signaling.
