EREG Human, HEK

What is EREG Human and what are its key biological functions?

EREG (Epiregulin) is part of the EGF family that functions as a ligand of EGFR (Epidermal Growth Factor Receptor) and most members of the ERBB family of tyrosine-kinase receptors. Human Epiregulin is initially synthesized as a glycosylated 19.0 kDa transmembrane precursor protein, which undergoes proteolytic cleavage to produce a 6.0 kDa mature secreted sequence .

From a functional perspective, EREG exhibits a dual regulatory role in cellular processes. It stimulates the proliferation of keratinocytes, hepatocytes, fibroblasts, and vascular smooth muscle cells while simultaneously inhibiting the growth of several tumor-derived epithelial cell lines . This bifunctional nature distinguishes EREG from other EGF family members and makes it particularly interesting for developmental biology and cancer research.

Why are HEK293 cells preferred for EREG production over other expression systems?

HEK293 cells offer several methodological advantages for EREG production compared to bacterial, yeast, or insect cell expression systems:

• Post-translational modifications: HEK293 cells perform human-specific glycosylation patterns and other post-translational modifications essential for EREG's biological activity

• Protein folding efficiency: These cells contain appropriate chaperone systems for proper folding of complex human proteins

• Secretion capability: HEK293 cells efficiently secrete recombinant proteins into the culture medium, simplifying purification

• Scalability: HEK cultures can be scaled up using suspension cultures for larger protein yields

• Transfection efficiency: HEK293 cells exhibit high transfection rates, making them suitable for both transient and stable expression

When specifically producing EREG Human Recombinant, HEK293 cells generate a properly folded, glycosylated polypeptide chain (63-108 a.a.) containing 289 amino acids with a molecular mass of 32.6 kDa . This mammalian expression system ensures the protein maintains its intended structure and function for research applications.

What are the standard quality control parameters for EREG Human produced in HEK cells?

The following quality control parameters should be assessed for EREG Human produced in HEK cells:

For storage stability, EREG Human should be stored at 4°C if using within 2-4 weeks, or frozen at -20°C for longer periods. Addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage, and multiple freeze-thaw cycles should be avoided to maintain protein integrity .

How can I optimize transient transfection protocols for maximum EREG yield in HEK293 cells?

Optimizing transient transfection for EREG production in HEK293 cells requires systematic adjustment of several parameters based on design of experiments (DoE) principles. Research has demonstrated that yields can be maximized by implementing the following methodological approach:

• Cell density optimization: Maintain cell density between 2.4-3.0 × 10^6 cells/mL at the time of transfection for optimal expression

• Transfection reagent concentration: When using polyethyleneimine (PEI), a concentration range of 24-30 mg/L provides optimal transfection efficiency while minimizing cytotoxicity

• DNA optimization:

  • Maintain plasmid DNA concentration around 1 mg/L, which limits the need for large DNA preparations while achieving good expression levels

  • For multi-chain proteins, maintain a 1:1 ratio of encoding plasmids

  • Use high-purity (endotoxin-free) plasmid preparations

• Culture conditions post-transfection:

  • Temperature shift to 32-34°C after initial 24h at 37°C can increase protein yields

  • Supplement with nutrient feed 24h post-transfection

  • Harvest secreted protein from culture supernatant 3-5 days post-transfection

This optimized protocol has been shown to be generally applicable to recombinant antibodies and proteins expressed in HEK cells , making it a suitable starting point for EREG production that can be further refined through iterative optimization.

What are the key differences in cellular responses when using EREG versus EGF in developmental organoid models?

EREG and EGF elicit fundamentally different cellular responses in developmental organoid models, particularly in intestinal enteroids. These differences provide critical insights into the specialized roles of these growth factors in tissue development:

These differences emerge even when EREG is used at concentrations (1-100 ng/mL) comparable to standard EGF concentrations in enteroid culture media . The remarkable ability of EREG to create spatially organized structures suggests it activates developmental programs that EGF does not, making it particularly valuable for creating more physiologically relevant organoid models for research.

How do EREG-induced signaling mechanisms differ across cell types, and what methodological considerations are important for comparative studies?

EREG-induced signaling exhibits significant cell type-specific variations that require careful methodological consideration when designing comparative studies:

• Receptor transactivation mechanisms:

  • In C9 hepatocytes and COS-7 cells, EREG signaling involves Src kinase and matrix metalloproteinase (MMP) dependent pathways

  • In HEK293 cells, signaling occurs independently of these mechanisms

  • These differences may reflect the absence of Src activation in certain cell types

• HB-EGF shedding dependency:

  • In some cell types, EREG signaling leads to MMP activation and subsequent shedding of heparin-binding EGF (HB-EGF)

  • This creates a secondary signaling cascade that amplifies the initial response

  • Blocking HB-EGF with neutralizing antibodies or CRM197 can attenuate downstream effects

• Receptor tyrosine involvement:

  • The role of specific tyrosine residues (such as Tyr319 in AT1-R) in EGF-R transactivation varies by cell type

  • Both wild-type and mutant (Y319F) AT1-R expressed in COS-7 cells can cause EGF-R transactivation through HB-EGF release

When designing comparative studies, researchers should implement these methodological controls:

• Use multiple cell lines with well-characterized receptor expression profiles

• Include pathway-specific inhibitors (Src inhibitors, MMP inhibitors)

• Perform both dose-response and time-course experiments to capture the full signaling dynamics

• Consider receptor expression levels when interpreting results

• Use specific blocking agents for potential intermediaries (HB-EGF neutralizing antibodies)

These considerations ensure that observed differences reflect genuine biological variation rather than technical artifacts.

How is EREG Human being utilized in advanced developmental biology and disease modeling applications?

EREG Human has emerged as a critical factor in developmental biology and disease modeling, particularly in intestinal organoid systems. Recent research has revealed several innovative applications:

• Developmental niche recreation: EREG has been identified as a growth factor robustly expressed in the stem cell domain of the developing human intestine . By incorporating EREG into enteroid culture media in place of the traditional EGF, researchers have successfully recreated the specialized developmental niche that supports proper intestinal epithelial organization.

• Advanced organoid engineering: EREG-grown enteroids develop with remarkable spatial organization that recapitulates native intestinal architecture. This includes properly formed crypt domains containing proliferative stem cells and a differentiated villus-like central lumen . This spatial organization has been difficult to achieve with conventional organoid culture methods.

• Rare cell population preservation: EREG-supplemented cultures maintain the full complement of epithelial cell types found in the native intestine, including difficult-to-preserve rare cell populations such as BEST4+ cells . This enables more comprehensive studies of specialized cell functions and rare disease phenotypes.

• Signal transduction modeling: EREG can be used to study differential signaling mechanisms across cell types, providing insights into cell-specific responses that may contribute to developmental disorders or cancer progression . The finding that EREG activates different signaling pathways in different cell types offers opportunities to model context-dependent cellular responses.

These applications demonstrate how EREG is advancing our ability to create more physiologically relevant models for studying human development and disease, particularly in epithelial tissues where spatial organization is crucial for function.

What role does EREG play in inflammatory responses and immune regulation?

Recent research has uncovered important connections between EREG signaling and immune system regulation:

• Inflammatory gene expression regulation: RNA-sequencing analysis of EREG-responsive systems has identified significant effects on genes related to interferon and cytokine signaling, including IRF5, NFKBIZ, and SOCS1 . Ingenuity analysis of these datasets revealed high statistical significance for inflammatory pathways including:

  • Granulocyte adhesion and diapedesis [-log(p value) = 12.8]

  • Role of cytokines in mediating communication between cells [-log(p value) = 9.86]

  • Differential regulation of cytokine production in macrophages and Th cells [-log(p value) = 6.89]

• Leukocyte recruitment and function: EREG expression is associated with biological processes including:

  • Accumulation of myeloid cells (p = 1.26E-17)

  • Accumulation of leukocytes (p = 6.75E-17)

  • Cytokine- and chemokine-mediated signaling pathways (p = 3.29E-16)

  • Accumulation and recruitment of granulocytes (p = 2.2E-14) and phagocytes (p = 1.76E-14)

• Expression pattern in immune tissues: EREG is expressed primarily in the placenta and peripheral blood leukocytes, suggesting immune-related functions . This expression pattern differs from other EGF family members and indicates potential specialized roles in immune homeostasis.

These findings suggest that EREG may serve as an important mediator in inflammatory responses and immune cell recruitment, making it a potential target for immunomodulatory interventions. The connection between EREG signaling and inflammatory gene expression also provides new avenues for understanding chronic inflammatory conditions and potential therapeutic approaches.

How can researchers accurately measure EREG activity and distinguish it from other EGF family members in complex biological systems?

Accurately measuring EREG activity in complex biological systems requires a multi-faceted approach that distinguishes its effects from other EGF family members:

• Receptor phosphorylation profiling:

  • Examine phosphorylation patterns of EGFR and related receptors (ERBB2-4)

  • EREG induces distinctive phosphorylation patterns of adapter molecules like Shc

  • Phospho-specific antibodies can detect these distinctive activation signatures

• Downstream signaling pathway analysis:

  • Monitor ERK1/2 and p90 ribosomal S6 kinase phosphorylation

  • Use specific pathway inhibitors to delineate EREG-dependent signaling components

  • Employ phospho-proteomics to create comprehensive signaling profiles

• Biological activity assessment:

  • Cell proliferation assays using Balb/3T3 mouse embryonic fibroblast cells (ED50 ≤ 1μg/ml for active EREG)

  • Spatial organization in enteroid cultures (EREG produces distinctive crypt-villus organization)

  • Cell type-specific responses (EREG inhibits certain tumor cell lines while stimulating others)

• Specific neutralization:

  • Use EREG-specific neutralizing antibodies to confirm activity attribution

  • Compare with pan-EGF family inhibitors to determine relative contribution

  • Include EGF receptor blocking antibodies as controls

• Gene expression analysis:

  • Identify EREG-specific transcriptional signatures through RNA-seq

  • Compare with signatures induced by other EGF family members

  • Focus on genes uniquely regulated by EREG signaling

These approaches, used in combination, allow researchers to specifically attribute biological effects to EREG rather than other EGF family members in complex systems such as tissue samples, patient-derived organoids, or in vivo models.

What are common challenges in producing high-quality EREG Human in HEK cells and how can they be addressed?

Researchers frequently encounter several challenges when producing EREG Human in HEK cells. The following table outlines these challenges and provides methodological solutions:

Additionally, researchers should implement systematic quality control testing throughout the production process, including SDS-PAGE for purity assessment, biological activity assays, and endotoxin testing to ensure consistent, high-quality EREG preparations suitable for sensitive research applications.

How do I design experiments to investigate differential effects of EREG versus EGF in cellular signaling pathways?

Designing experiments to investigate differential effects of EREG versus EGF requires careful consideration of multiple variables to ensure robust and interpretable results:

• Experimental design framework:

  • Include matched concentrations of EREG and EGF (1-100 ng/mL range)

  • Perform complete dose-response curves to identify potential potency differences

  • Include time-course analyses (5 min to 24 h) to capture transient and sustained signaling events

  • Compare multiple cell types with characterized receptor expression profiles

• Critical control experiments:

  • Receptor expression analysis: Quantify EGFR and ERBB2-4 levels in test cells

  • Pathway-specific positive controls: Include known activators of MAPK, PI3K, and other relevant pathways

  • Negative controls: Include receptor-specific inhibitors (e.g., gefitinib for EGFR)

  • Cellular context controls: Test effects in both 2D and 3D culture systems

• Methodological approaches for pathway analysis:

  • Phosphorylation status: Measure receptor and downstream effector (ERK1/2, AKT) phosphorylation

  • Adapter recruitment: Assess Shc phosphorylation and complex formation

  • Gene expression: Perform RNA-seq to identify pathway-specific transcriptional signatures

  • Proteomics: Use phospho-proteomics to create comprehensive signaling profiles

• Cell type-specific considerations:

  • Test in multiple cell lines (e.g., C9 hepatocytes, COS-7, HEK293)

  • Include cells with different dependencies on Src kinase and MMP activity

  • Consider the role of HB-EGF shedding in signal transduction

  • Test impacts of matrix and cell-cell contacts in epithelial models

This experimental design approach will allow researchers to distinguish the unique signaling properties of EREG from those of EGF across different cellular contexts, providing insights into their distinct biological roles.

What specific considerations are necessary when using EREG in specialized research applications like organoid development?

When incorporating EREG into specialized research applications like organoid development, researchers should consider several critical methodological factors:

• Concentration optimization:

  • Test multiple concentrations (1 ng/mL, 10 ng/mL, and 100 ng/mL) to determine optimal effects

  • Document concentration-dependent morphological and functional outcomes

  • Consider that optimal concentrations may differ between developmental stages and tissue types

• Media formulation compatibility:

  • EREG must be used in the context of appropriate base media formulation

  • Confirm compatibility with other critical growth factors (e.g., Wnt, R-spondin, Noggin)

  • Test for potential synergistic or antagonistic interactions with other media components

• Temporal considerations:

  • Allow sufficient time (10+ days) for spatial organization to develop in EREG-supplemented cultures

  • Document the temporal progression of morphological changes

  • Consider stage-specific requirements that may necessitate sequential media adjustments

• Validation approaches:

  • Compare morphology between EREG-grown and EGF-grown structures using brightfield microscopy

  • Perform immunostaining for domain-specific markers to confirm spatial organization

  • Use single-cell RNA sequencing (scRNA-Seq) to verify appropriate cell type composition

  • Validate functional properties specific to the organoid type (e.g., barrier function, secretion)

• Specialized applications:

  • For developmental biology: Match EREG concentration to developmental stage being modeled

  • For disease modeling: Consider disease-specific alterations in EREG signaling

  • For drug screening: Standardize culture conditions to minimize variability

• Quality control metrics:

  • Define objective criteria for successful organoid development (budding frequency, size distribution)

  • Implement quantitative image analysis to ensure consistency

  • Establish molecular and functional benchmarks for quality assessment

These considerations will help researchers successfully implement EREG in specialized applications, particularly those requiring proper spatial organization and cellular differentiation, such as advanced intestinal organoid models for developmental biology and disease research.

What emerging research opportunities exist for EREG Human in disease modeling and therapeutic development?

EREG Human presents several promising research opportunities at the intersection of disease modeling and therapeutic development:

• Advanced cancer modeling:

  • EREG inhibits the growth of several tumor-derived epithelial cell lines , suggesting potential anti-cancer applications

  • The dual stimulatory/inhibitory nature of EREG could be exploited for targeted therapy approaches

  • EREG expression in specific carcinomas of the bladder, lung, kidney, and colon indicates potential as a biomarker

• Spatially organized disease models:

  • EREG's ability to create spatially organized enteroids enables more accurate modeling of diseases affecting specific intestinal compartments

  • This provides platforms for:

    • Inflammatory bowel disease research with proper crypt-villus organization

    • Colorectal cancer progression models with defined compartmentalization

    • Developmental disorder investigations requiring proper tissue architecture

• Immune modulation approaches:

  • EREG's expression in peripheral blood leukocytes and involvement in multiple immune-related pathways suggests immunomodulatory potential

  • Research opportunities include:

    • Targeting EREG-dependent inflammatory pathways in chronic inflammatory conditions

    • Exploiting EREG's role in leukocyte recruitment for immunotherapy approaches

    • Investigating EREG in viral resistance mechanisms

• Regenerative medicine applications:

  • EREG stimulates proliferation of multiple cell types including keratinocytes, hepatocytes, and fibroblasts

  • This proliferative capacity could be harnessed for:

    • Wound healing and tissue regeneration therapies

    • Engineering of complex tissue grafts with proper spatial organization

    • Ex vivo expansion of primary tissue for transplantation

These research directions leverage EREG's unique properties to address unmet needs in disease modeling and therapeutic development, particularly where spatial organization and immune modulation are critical factors.

How might developments in cell signaling research impact our understanding of EREG's role in development and disease?

Emerging developments in cell signaling research are poised to transform our understanding of EREG's biological roles through several mechanisms:

• Receptor heterodimer specificity:

  • Advanced imaging techniques now allow visualization of receptor dimer formation in real-time

  • This may reveal EREG's preference for specific EGFR/ERBB family heterodimer combinations

  • Such preferences could explain cell type-specific responses and differential effects compared to EGF

  • Understanding these preferences could clarify EREG's unique developmental roles

• Signaling dynamics and computational modeling:

  • New approaches in signaling dynamics reveal that temporal patterns of pathway activation are as important as magnitude

  • EREG may induce distinctive temporal signaling signatures different from other EGF family members

  • Computational models integrating these dynamics could predict cell-specific responses to EREG

  • Such models may explain the remarkable spatial organization observed in EREG-grown enteroids

• Single-cell signaling heterogeneity:

  • Single-cell analysis techniques have revealed substantial cell-to-cell variability in signaling responses

  • EREG may induce different response patterns across cell populations

  • This heterogeneity could drive important developmental processes requiring cellular diversity

  • Understanding this dimension may clarify EREG's role in creating organized tissue structures

• Integration with mechanobiology:

  • Emerging research shows bidirectional relationships between mechanical forces and growth factor signaling

  • EREG signaling may be modulated by tissue mechanics differently than other growth factors

  • This interaction could explain EREG's unique effects in three-dimensional tissue contexts

  • Mechanistic insights could lead to improved organoid culture systems with more physiological organization

These advancing frontiers in signaling research will likely provide deeper insights into how EREG's distinctive signaling properties contribute to its specialized roles in development, homeostasis, and disease.

What methodological advances are needed to better characterize the unique properties of EREG compared to other EGF family members?

Several methodological advances are needed to more comprehensively characterize EREG's unique properties:

• Advanced receptor binding studies:

  • Development of EREG-specific fluorescent probes for real-time binding visualization

  • Single-molecule tracking to determine receptor clustering and internalization dynamics

  • Quantitative binding assays across the full ERBB receptor family with comparative kinetics

  • These approaches would provide direct evidence of EREG's binding preferences and kinetics

• Spatiotemporal signaling analysis:

  • Live-cell biosensors for real-time visualization of EREG-induced signaling events

  • Multi-parametric signaling analysis at single-cell resolution

  • Advanced phospho-proteomics with high temporal resolution to capture signaling dynamics

  • Integration of these datasets would reveal EREG's unique signaling "fingerprint"

• Structural biology approaches:

  • High-resolution crystal structures of EREG-receptor complexes

  • Comparative structural analysis with other EGF family member-receptor complexes

  • Molecular dynamics simulations to predict ligand-specific conformational changes

  • These structural insights would explain mechanistic differences in receptor activation

• Improved organoid analysis techniques:

  • Automated image analysis platforms for quantifying spatial organization in 3D cultures

  • Live imaging of developing organoids to track morphogenesis in real-time

  • Spatial transcriptomics to map gene expression patterns across organoid domains

  • Such techniques would provide quantitative metrics of EREG's unique organizational effects

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Network analysis to identify EREG-specific signaling hubs and feedback mechanisms

  • Comparative pathway analysis across multiple EGF family members

  • These integrative approaches would reveal emergent properties of EREG signaling networks

These methodological advances would move beyond the current descriptive understanding of EREG's effects to provide mechanistic insights into its unique properties and biological roles, particularly in complex developmental contexts.