EPCAM Human, sf9

What is the structure of human EpCAM and how does it function in normal and cancer tissues?

EpCAM is a transmembrane glycoprotein that undergoes regulated intramembrane proteolysis to generate functionally active fragments. Structurally, it consists of an N-terminal extracellular domain (EpEX), a single transmembrane domain, and a short C-terminal intracellular domain (EpICD).

In normal tissues, EpCAM shows tissue-specific expression patterns. It is strongly expressed in epithelial cells of various organs but notably absent in hepatocytes, blood vessels, muscle, fat, lymph nodes, spleen, and brain tissues . Specific expression patterns include:

• Strong expression in distal tubuli of kidney

• Moderate expression in spermatogonia and spermatocytes

• Expression in peripheral germinative cells of sebaceous glands and eccrine glands

• Strong expression in urothelium

• Strong expression in luminal cells of breast glands with reduced or absent staining in myoepithelial cells

In cancer tissues, EpCAM serves as a carcinoma marker and is differentially expressed across tumor types. For example, it shows high expression in basal cell carcinoma (81.1% strong positivity) and Merkel cell carcinoma (74.4% strong positivity), while being completely absent in malignant melanoma .

Why is the Sf9 insect cell system preferred for expressing human EpCAM protein?

The Sf9 insect cell expression system offers several methodological advantages for EpCAM production:

• Post-translational modifications: Sf9 cells can perform many eukaryotic post-translational modifications that bacterial systems cannot, allowing for proper folding and glycosylation of EpCAM.

• Higher yield: The baculovirus-Sf9 system typically produces higher quantities of recombinant protein compared to mammalian systems.

• Structural integrity: The system facilitates proper formation of disulfide bonds critical for EpCAM's structural domains, particularly in the thyroglobulin domain and C-terminal domain that form extensive interactions in the functional dimer .

• Scalability: Sf9 cultures can be readily scaled up for protein production without losing expression efficiency.

• Protein solubility: The system often yields soluble protein, which is essential for structural studies and functional assays of EpCAM.

How can I verify the correct expression and folding of recombinant human EpCAM in Sf9 cells?

Verification of proper EpCAM expression and folding requires multiple analytical approaches:

• Western blotting: Using antibodies specific to different epitopes of EpCAM (e.g., Ber-EP4 or MSVA-326R antibodies mentioned in the literature ).

• Dimer formation analysis: Native PAGE or size exclusion chromatography to verify the formation of EpCAM dimers, which are critical for function.

• Glycosylation assessment: Peptide-N-glycosidase F (PNGase F) treatment followed by SDS-PAGE to assess N-glycosylation status.

• Functional binding assays: Testing interaction with known binding partners such as EGFR to confirm proper folding and function .

• Structural validation: Circular dichroism spectroscopy to assess secondary structure content, which should match the expected profile of properly folded EpCAM.

What experimental approaches can be used to study EpCAM dimer stability and how do specific mutations affect it?

The stability of EpCAM dimers can be studied using several approaches:

• Molecular dynamics simulations: As demonstrated in the literature, all-atom molecular dynamics simulations (5 ns) can be used to analyze inter-residue contacts between EpCAM subunits and evaluate dimer stability . This approach requires:

  • Preparation of topology files using software like VMD

  • System solvation with appropriate water margin (20Å)

  • System neutralization with sodium or chloride ions

  • Equilibration at physiological conditions (310K, 1 atm)

  • Analysis of trajectory files to obtain inter-residue distance vs. time plots

• Mutagenesis studies: Strategic mutations can be introduced to either stabilize or destabilize the EpCAM dimer. Key residues identified for mutagenesis include:

  • Residues in the thyroglobulin domain loop (TYloop): Arg80, Arg81, Lys83

  • Residues in the C-terminal domain β-sheet: Glu147, Glu187, Asp194

  • Additional residues identified through MD simulations: Leu88, Pro84

• Inter-residue contact network analysis: Using tools like UCSF Chimera with Cytoscape to visualize and quantify dimer interface interactions.

Specific mutations shown to destabilize the dimer include K83D, P84D, and L88D, which disrupt critical interactions at the dimer interface . When expressing these mutant forms in Sf9 cells, researchers should expect altered dimer formation kinetics that can be quantified using analytical techniques like size exclusion chromatography or native PAGE.

How does EpCAM interact with EGFR signaling pathways, and what methodologies can be used to study this interaction in recombinant systems?

EpCAM and EGFR exhibit significant molecular cross-talk that affects cancer cell proliferation and EMT. The extracellular domain of EpCAM (EpEX) functions as a ligand for EGFR with distinct effects compared to EGF .

Methodological approaches to study this interaction include:

• Co-immunoprecipitation assays: Using recombinant EpEX from Sf9 cells to pull down EGFR from cell lysates.

• Surface plasmon resonance (SPR): To determine binding kinetics and affinity between EpEX and EGFR.

• Phosphorylation studies: Measuring EGFR downstream signaling activation (pERK1/2, pAKT) after treatment with purified EpEX using Western blotting .

• Competitive binding assays: Determining if EpEX competes with EGF for EGFR binding.

• Functional cellular assays: Assessing the effects of purified EpEX on:

  • Cell proliferation

  • EMT marker expression

  • Cell migration

  • Expression of EMT transcription factors (Snail, Zeb1, Slug)

Notably, EpEX activates pERK1/2 and pAKT to induce EGFR-dependent proliferation while repressing EGF-mediated EMT . When designing experiments with recombinant EpEX from Sf9 cells, researchers should include appropriate controls to distinguish between EpEX and EGF effects on EGFR signaling.

What are the critical quality control parameters for recombinant EpCAM produced in Sf9 cells, and how do they affect experimental outcomes?

Quality control of Sf9-expressed EpCAM requires rigorous assessment of multiple parameters:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (>95% purity recommended)

  • Mass spectrometry to confirm protein identity and detect potential post-translational modifications

• Structural integrity:

  • Circular dichroism to confirm secondary structure

  • Thermal shift assays to assess protein stability

  • Size exclusion chromatography to evaluate oligomeric state

• Functional validation:

  • Binding assays with known interaction partners (EGFR, claudins, CD44, E-cadherin)

  • Cell adhesion assays if using EpCAM for cell-cell interaction studies

• Glycosylation analysis:

  • Lectin blotting to characterize glycan composition

  • Mass spectrometry to identify glycosylation sites and patterns

  • Comparison with native human EpCAM glycosylation profile

• Endotoxin testing:

  • Limulus amebocyte lysate (LAL) assay to ensure preparations are endotoxin-free (<0.1 EU/mg protein)

Each parameter directly impacts experimental outcomes. For instance, incompletely processed EpCAM may not form proper dimers, affecting binding studies. Similarly, aberrant glycosylation can alter protein-protein interactions and immunogenicity in functional assays.

How can I design experiments to evaluate the differential effects of full-length EpCAM versus its proteolytic fragments produced in Sf9 cells?

Designing experiments to compare full-length EpCAM with its proteolytic fragments requires careful planning:

• Expression strategy:

  • Full-length EpCAM: Express with appropriate signal sequence for membrane localization

  • EpEX (extracellular domain): Express with C-terminal truncation before the transmembrane domain

  • EpICD (intracellular domain): Express with appropriate tags for detection and purification

• Purification considerations:

  • Full-length EpCAM requires detergent solubilization from membranes

  • EpEX can be secreted into the medium

  • EpICD requires specific buffer conditions to maintain solubility

• Functional comparison assays:

  • Signaling activation: Compare effects on WNT and Ras/Raf pathway components

  • Protein-protein interactions: Evaluate differential binding to partners like claudins, CD44, E-cadherin, and EGFR

  • Subcellular localization: Track distribution using fluorescently tagged constructs

  • Transcriptional effects: Measure impact on gene expression profiles, particularly those related to proliferation and EMT

• Controls to include:

  • Enzymatically generated fragments from full-length protein for comparison with recombinantly expressed fragments

  • Mutants that cannot undergo proteolytic processing

  • Concentration-matched samples to ensure comparable molar quantities

What are common expression issues when producing human EpCAM in Sf9 cells and how can they be addressed?

Several challenges can arise during EpCAM expression in Sf9 cells:

• Low expression yield:

  • Solution: Optimize codon usage for Sf9 cells

  • Adjust MOI (multiplicity of infection) and harvest time

  • Test different signal sequences for improved secretion

  • Evaluate multiple cell lines (Sf9, Sf21, High Five™)

• Protein aggregation:

  • Solution: Modify buffer conditions (pH, salt concentration)

  • Add stabilizing agents (glycerol, sucrose)

  • Reduce expression temperature to 27°C

  • Consider fusion partners that enhance solubility

• Incomplete post-translational modifications:

  • Solution: Ensure sufficient expression time for complete processing

  • Supplement culture medium with necessary precursors

  • Consider mammalian expression for complex glycosylation if required

• Proteolytic degradation:

  • Solution: Add protease inhibitors during purification

  • Remove protease-sensitive site Gly79-Arg80-Arg81 while maintaining protein functionality

  • Purify at lower temperatures (4°C)

• Improper folding:

  • Solution: Include molecular chaperones in expression system

  • Add stabilizing ligands during expression

  • Implement step-wise refolding protocols if necessary

How can intratumoral heterogeneity of EpCAM expression be accurately modeled using recombinant systems?

Modeling intratumoral heterogeneity of EpCAM expression requires sophisticated experimental approaches:

• Generate expression variants:

  • Create multiple EpCAM constructs reflecting tumor-specific mutations

  • Express EpCAM variants with different post-translational modifications

  • Produce both full-length and naturally occurring truncated forms

• Develop mixed population assays:

  • Combine different ratios of EpCAM variants to mimic heterogeneous expression

  • Create gradient systems with variable EpCAM density

  • Establish co-culture systems with variable EpCAM-expressing cells

• Implement quantitative analysis:

  • Use flow cytometry to quantify expression level distributions

  • Apply high-content imaging to assess spatial heterogeneity

  • Develop ELISA or other quantitative assays calibrated to known EpCAM concentrations

• Correlation with clinical data:

  • Design experiments that reflect the heterogeneity observed in specific tumor types (Table 1)

Table 1: EpCAM Expression Heterogeneity in Selected Tumor Types

Data adapted from EpCAM immunostaining results

How can recombinant EpCAM be utilized to develop improved circulating tumor cell (CTC) detection methods?

Recombinant EpCAM from Sf9 cells can advance CTC detection methods through:

• Antibody development and validation:

  • Generate high-affinity antibodies against recombinant EpCAM

  • Use recombinant protein for epitope mapping

  • Validate antibody specificity against different EpCAM conformations and fragments

  • Develop sandwich ELISA formats with optimized antibody pairs

• Microfluidic capture device optimization:

  • Coat devices with recombinant EpCAM antibodies at controlled densities

  • Test binding kinetics under flow conditions

  • Optimize buffer conditions to maximize specific binding while minimizing non-specific interactions

• Quality control standards:

  • Develop calibration curves using known amounts of recombinant EpCAM

  • Create artificial CTCs by coating microbeads with recombinant EpCAM at defined densities

  • Establish positive controls for CTC enrichment systems

• Heterogeneity assessment:

  • Develop detection systems that account for variable EpCAM expression in CTCs

  • Create assays capable of detecting EpCAM-low cells that would be missed by conventional approaches

• Clinical validation:

  • Correlate CTC counts with patient outcomes as demonstrated in clinical studies, where patients with ≥5 CTCs showed significantly poorer prognosis

What are the critical considerations when designing structure-function studies of EpCAM using protein expressed in Sf9 cells?

Structure-function studies of EpCAM require careful experimental design:

• Domain-specific analysis:

  • Express individual domains (thyroglobulin-like domain, C-terminal domain) to study their specific functions

  • Create domain-swapping constructs to identify functional regions

  • Generate point mutations in key residues identified through structural analysis

• Dimer interface investigation:

  • Focus on the TYloop (thyroglobulin domain loop) that forms extensive interactions with the β-sheet in the C-terminal domain of the juxtaposed subunit

  • Pay special attention to electrostatic interactions between Arg80, Arg81, and Lys83 in the TYloop and the negatively charged patch formed by Glu147, Glu187, Asp194 in the β-sheet

  • Design mutations that either stabilize or destabilize the dimer without affecting domain folding

• Functional readouts:

  • Cell adhesion assays to evaluate homophilic binding

  • EGFR signaling assays to assess EpEX functionality

  • WNT pathway activation to measure EpICD activity

  • EMT marker expression to evaluate regulatory functions

• Structural validation technologies:

  • X-ray crystallography for high-resolution structure determination

  • Cryo-EM for visualizing larger complexes

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) to identify dynamic regions and binding interfaces

  • SAXS (small-angle X-ray scattering) for solution structure analysis

• Controls and standards:

  • Compare with mammalian-expressed EpCAM to account for expression system-specific differences

  • Include clinically relevant mutations identified in patient samples

How can recombinant EpCAM contribute to the development of targeted therapies, including CAR-T approaches?

Recombinant EpCAM from Sf9 cells can accelerate therapeutic development through:

• Epitope mapping for therapeutic antibodies:

  • Identify optimal binding sites that affect EpCAM function

  • Screen antibody libraries against recombinant EpCAM

  • Evaluate antibody-EpCAM complexes using structural biology approaches

  • Test antibody efficacy in blocking EpCAM-mediated signaling

• CAR-T cell optimization:

  • Use recombinant EpCAM to select optimal single-chain variable fragments (scFvs)

  • Evaluate CAR binding affinity and specificity

  • Develop assays to predict potential on-target/off-tumor effects based on EpCAM expression patterns in normal tissues

  • Optimize CAR-T activation thresholds using recombinant EpCAM at varying densities

• Bispecific antibody development:

  • Create and test EpCAM-targeting arms for bispecific constructs

  • Evaluate dual targeting strategies (e.g., EpCAM-EGFR) based on known pathway interactions

  • Optimize binding kinetics for maximal therapeutic effect

• Drug conjugate testing:

  • Use recombinant EpCAM to evaluate antibody-drug conjugate binding and internalization

  • Develop assays for measuring drug release kinetics after EpCAM binding

  • Test stability of conjugates under physiological conditions

• Patient stratification tools:

  • Develop diagnostics to identify patients likely to benefit from EpCAM-targeted therapies

  • Create assays to detect EpCAM variants that might confer resistance to targeted therapies

What emerging approaches can help resolve discrepancies in EpCAM expression data across different tumor types?

The literature shows significant discrepancies in reported EpCAM expression across tumor types, with positivity ranging from 0-100% in hepatocellular carcinoma, 0-54% in epithelioid mesothelioma, and 17-100% in breast cancers . Emerging approaches to resolve these inconsistencies include:

• Standardized detection protocols:

  • Develop reference standards using recombinant EpCAM from Sf9 cells

  • Create calibration curves for quantitative IHC/IF

  • Establish international proficiency testing programs

• Multi-epitope detection strategies:

  • Use antibody panels targeting different EpCAM domains

  • Compare antibodies like MSVA-326R and Ber-EP4 systematically

  • Develop RNA-protein correlation studies to address discrepancies

• Single-cell analysis technologies:

  • Apply single-cell proteomics to quantify EpCAM at individual cell level

  • Combine with spatial transcriptomics to map heterogeneity

  • Correlate with functional markers of EMT and stemness

• Digital pathology and AI approaches:

  • Implement machine learning algorithms for standardized scoring

  • Develop quantitative intensity metrics independent of observer bias

  • Create digital reference atlases of EpCAM expression

• Temporal dynamics studies:

  • Establish methods to track EpCAM expression changes over time

  • Develop live-cell imaging approaches with EpCAM reporters

  • Create patient-derived models that maintain tumor heterogeneity

How might structural studies of EpCAM expressed in Sf9 cells advance our understanding of its role in epithelial-to-mesenchymal transition?

Structural studies using Sf9-expressed EpCAM can provide crucial insights into EMT regulation:

• Structure-based investigation of EpCAM-EGFR interaction:

  • Determine the crystal structure of EpEX-EGFR complex

  • Map binding interfaces that distinguish EpEX from EGF in EGFR activation

  • Identify structural elements responsible for differential activation of ERK1/2 signaling

• Conformational dynamics during proteolytic processing:

  • Study structural changes that occur during regulated intramembrane proteolysis

  • Identify conformational switches that regulate release of EpICD

  • Map structural determinants of protease recognition sites

• Interaction networks with EMT regulators:

  • Characterize structural basis of interactions with E-cadherin

  • Map binding interfaces with EMT transcription factors

  • Investigate potential direct or indirect interactions with β-catenin

• High-resolution structure of full-length EpCAM:

  • Use cryo-EM to solve structure of membrane-embedded EpCAM

  • Identify conformational changes associated with signaling

  • Map lipid interactions that may regulate function

• Dynamic structural changes during EMT:

  • Develop structural probes to monitor EpCAM conformational states

  • Create biosensors based on EpCAM structural features to monitor EMT progression

  • Identify potential druggable pockets that emerge during conformational changes

By combining these structural approaches with functional studies, researchers can develop a mechanistic understanding of how EpCAM dynamically regulates the balance between epithelial maintenance and EMT induction, potentially leading to new therapeutic strategies targeting specific conformational states of EpCAM in cancer.