EPHA2 Human, sf9
Description
Production and Purification
Key Steps:
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Expression: Optimized in Sf9 cells to ensure proper folding and stability, unlike bacterial systems (e.g., E. coli) .
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Purification: Proprietary chromatographic techniques yield >95% purity (SDS-PAGE verified) .
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Labeling: Uniform 15N-amino acid labeling in Sf9 enables NMR studies of dynamics and drug interactions .
Stability:
Role in Cancer:
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Overexpressed in breast, head/neck, and non-small-cell lung cancers .
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Binds ephrin-A ligands (e.g., ephrinA1) to regulate pathways like ERK and AKT .
Key Research Findings:
Applications in Biomedical Research
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Drug Development: Used in high-throughput screening for kinase inhibitors .
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Structural Biology: Facilitates X-ray crystallography and NMR studies of receptor-ligand interactions .
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Cancer Therapeutics: EphA2 downregulation reverses malignant traits (e.g., invasion in glioblastoma) .
Comparative Advantages Over Other Systems
Product Specs
Q&A
Table 1: EPHA2 Expression Systems Comparison
| Parameter | Sf9 Insect Cells | HEK293 Mammalian Cells |
|---|---|---|
| Yield | 2–5 mg/L | 0.5–1 mg/L |
| Glycosylation | Paucimannose-type | Complex N-linked |
| Phosphorylation | Limited | Native-like |
| Typical Use Case | Structural studies | Signaling pathway analysis |
How do I validate EPHA2 ligand-binding activity in Sf9-expressed protein?
Ligand-binding validation requires a multi-modal approach:
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Surface Plasmon Resonance (SPR): Immobilize EPHA2 on a CM5 biosensor chip and measure kinetics with ephrinA1 or ephrinA5 ligands. For example, studies report a K<sub>D</sub> of 120–450 nM for ephrinA1-EPHA2 interactions using SPR .
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Crystallography: Resolve EPHA2-ephrin complexes to identify critical interfaces (e.g., the "head–tail" asymmetric interactions in ligand-free states) .
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Cell-based assays: Co-culture Sf9-expressed EPHA2 with ephrin-transfected mammalian cells to monitor receptor clustering via fluorescence cross-correlation spectroscopy (FCCS) .
Why does Sf9-expressed EPHA2 exhibit variable phosphorylation in kinase assays?
EPHA2 autophosphorylation in Sf9 systems is context-dependent. Ligand-free EPHA2 primarily shows phosphorylation at S897 (non-canonical pathway), while ephrin-bound states activate canonical Y772 phosphorylation . To resolve variability:
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Pre-treat cells with phosphatase inhibitors (e.g., sodium orthovanadate).
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Use ATP-competitive inhibitors (e.g., dasatinib) to distinguish baseline vs. ligand-induced activity .
How to resolve contradictory data on EPHA2’s dual oncogenic/tumor-suppressive roles?
EPHA2’s functional duality arises from ligand availability and cellular context. For example:
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Ligand-dependent signaling: EphrinA1 binding induces receptor clustering and Y772 phosphorylation, suppressing RAS-MAPK pathways in glioblastoma .
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Ligand-independent signaling: In Sf9 systems, unliganded EPHA2 forms head–tail (HT) oligomers that activate pro-migratory S897 phosphorylation via AKT .
Experimental Design Recommendation:
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Model selection: Use Sf9 systems for structural studies of HT interactions and mammalian models (e.g., HEK293T) for pathway analysis.
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Contextual controls: Compare EPHA2 mutants (e.g., S897A vs. Y772F) in invasion assays to isolate signaling nodes .
What methodologies identify EPHA2 clustering mechanisms in Sf9 membranes?
EPHA2 clustering is driven by ectodomain interfaces:
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PIE-FCCS: Resolve real-time oligomerization using pulsed interleaved excitation fluorescence cross-correlation spectroscopy. Studies show that unliganded EPHA2 forms linear arrays via conserved LBD-sushi domain interfaces .
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Cryo-EM: Resolve extended EphA2-ephrin assemblies (e.g., 4.2 Å structures of eEphA2-ephrinA5RBD complexes) .
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Mutagenesis: Disrupt interfaces B (LBD-LBD) and D (sushi-sushi) to abrogate clustering. For example, HEK293T cells expressing EphA2<sup>D758A</sup> show reduced cell-cell contact localization .
How to address EPHA2 degradation artifacts in Sf9 purification workflows?
EPHA2 undergoes activation-dependent proteolysis, producing ~70–95 kDa fragments . Mitigation strategies include:
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Protease inhibitors: Use leupeptin (10 μM) and pepstatin A (1 μM) during lysis.
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Temperature optimization: Maintain cultures at 27°C (≥30°C increases caspase-like protease activity).
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Affinity tags: Use C-terminal His-tags to isolate full-length protein via Ni-NTA chromatography .
What computational tools predict EPHA2 interactomes in Sf9 vs. human cells?
Product Science Overview
EPHA2 is a glycosylated protein that consists of an extracellular region, a single transmembrane segment, and a cytoplasmic tyrosine kinase domain. The extracellular region contains a ligand-binding domain, a cysteine-rich domain, and two fibronectin type III repeats. The cytoplasmic region includes a juxtamembrane segment, a tyrosine kinase domain, and a sterile alpha motif (SAM) domain.
The recombinant human EPHA2 protein expressed in Sf9 cells (a cell line derived from the fall armyworm, Spodoptera frugiperda) is produced using a baculovirus expression system. This system is advantageous for producing high yields of recombinant proteins with post-translational modifications similar to those in mammalian cells .
EPHA2 interacts with ephrin-A family ligands, which are membrane-bound proteins on adjacent cells. This interaction triggers bidirectional signaling: forward signaling through the Eph receptor and reverse signaling through the ephrin ligand. These signaling pathways regulate various cellular processes:
- Cell Migration and Adhesion: EPHA2 activation influences integrin-mediated adhesion and cell migration, which are critical for tissue development and repair.
- Cell Proliferation and Differentiation: EPHA2 signaling can either promote or inhibit cell proliferation and differentiation, depending on the cellular context.
- Tumor Growth and Metastasis: In cancer, EPHA2 is often overexpressed and associated with increased tumor growth, angiogenesis, and metastasis. It can also contribute to resistance to certain therapies .
Recombinant EPHA2 protein is widely used in research to study its role in various biological processes and diseases. It is particularly valuable in:
- Cancer Research: Understanding the mechanisms by which EPHA2 contributes to tumor progression and metastasis can lead to the development of targeted therapies.
- Drug Screening: Recombinant EPHA2 is used in high-throughput screening assays to identify potential inhibitors that can block its activity.
- Structural Studies: Analyzing the structure of EPHA2 helps in understanding its interaction with ligands and other proteins, which is crucial for designing effective drugs .
