FASLG Human, HEK

What is human FASLG and what experimental systems are used to study it?

Human Fas ligand (FasL, also known as TNFSF6, CD95L, or CD178) is a type II transmembrane protein belonging to the TNF superfamily. It plays a crucial role in immune system regulation by inducing apoptosis in target cells through binding to the Fas receptor (CD95/APO-1/TNFRSF6). FASLG is primarily expressed on activated T-cells, NK cells, and other immune cells .

For experimental studies, researchers commonly use:

• Recombinant extracellular domains fused to tags (e.g., Fc chimeras)

• HEK 293 cell expression systems for protein production

• Jurkat T cells for functional studies of Fas/FasL signaling

The extracellular domain of human Fas ligand (hFasLECD) is particularly relevant for research as it retains binding activity to Fas receptor and can be more easily manipulated in experimental settings .

Why are HEK 293 cells preferred for recombinant human FASLG production?

HEK 293 cells are widely used for recombinant FASLG production due to several key advantages:

• Protein folding and post-translational modifications: HEK cells provide human-like glycosylation patterns and proper protein folding machinery, essential for maintaining FASLG's native structure and function .

• Expression efficiency: These cells offer high transfection efficiency and protein yield.

• Secretion capability: HEK cells efficiently secrete recombinant proteins into the culture medium, facilitating purification processes .

• Validated protocols: Well-established methods exist for producing FASLG in HEK cells, including fusion to the Fc portion of human IgG1 to enhance stability and purification .

HEK-expressed FASLG demonstrates reliable biological activity in functional assays, making it suitable for studying Fas/FasL interactions and downstream signaling pathways .

What are the structural characteristics of human FASLG that impact experimental design?

Understanding the structural features of human FASLG is critical for experimental design:

• Domain structure: Human FASLG consists of an N-terminal cytoplasmic domain (1-80), transmembrane region (81-102), and C-terminal extracellular domain (103-281) .

• Functional oligomerization: FASLG functions as a homotrimer, and this oligomeric state is crucial for its biological activity .

• Soluble vs. membrane-bound forms: Both membrane-bound and proteolytically cleaved soluble forms exist, with different biological activities .

For research applications, engineered forms often include:

• Extracellular domain fusions with Fc (aa 7-154 of Fas fused to Fc)

• FLAG-tagged versions with isoleucine zipper motifs for self-oligomerization (FasL-LZ)

• PAGFP and PAmCherry fusion constructs for imaging studies

These structural considerations are essential when designing experiments to study FASLG-mediated signaling and cellular responses.

How can I optimize expression and purification of bioactive recombinant human FASLG in HEK cells?

Optimizing FASLG expression and purification requires attention to several critical factors:

Expression optimization:

• Vector selection: Use vectors with strong promoters (e.g., CMV) and appropriate signal sequences for secretion .

• Cell density and viability: Maintain HEK 293 cells at 1-2 × 10^7 cells/mL with >95% viability before transfection .

• Transfection method: Electroporation (e.g., BTX ECM 630 system) or lipid-based transfection can be used depending on scale .

• Expression time: Collect supernatants 24-72 hours post-transfection, balancing protein yield with quality .

Purification strategy:

• Affinity purification: Use anti-FLAG M2 beads for FLAG-tagged constructs or Protein A/G for Fc-fusion proteins .

• Quantification: ELISA with standard curves for accurate determination of protein concentration .

• Quality control: Assess purity by SDS-PAGE (≥95%) and biological activity through functional assays .

Storage and stability:

• Store purified protein in aliquots at -20°C to avoid freeze-thaw cycles .

• Reconstituted protein remains stable for at least 6 months when properly stored .

Following these guidelines will help ensure consistent production of functional FASLG proteins for experimental applications.

What imaging techniques are most effective for studying FASLG-Fas receptor interactions?

Several advanced imaging techniques have been developed to visualize FASLG-Fas receptor interactions:

Super-resolution imaging approaches:

• Structured Illumination Microscopy (SIM): Enables visualization of receptor reorganization following FASLG stimulation with resolution beyond the diffraction limit. This technique has been successfully used with Fas-Emerald GFP, FADD-mCherry, and caspase-8-TagRFP fusion constructs .

• Photoactivated Localization Microscopy (PALM): Provides nanometer-scale resolution of receptor clustering using photoactivatable fluorescent proteins such as PAGFP and PAmCherry .

Experimental setup for live-cell imaging:

• Transfect cells with appropriate fusion constructs 24 hours before imaging .

• For stimulation experiments, use FasL-LZ (extracellular domain of Fas ligand fused to a FLAG-tag and isoleucine zipper motif) .

• Employ flow chambers with supported lipid bilayers for controlled stimulation .

Fluorescence resonance energy transfer (FRET):

• Use mCerulean3 and mVenus-tagged constructs for quantitative measurements of protein-protein interactions in the Fas signaling pathway .

• Flow cytometric FRET provides population-level analysis of interaction dynamics .

These imaging approaches offer complementary information about spatial organization, temporal dynamics, and molecular interactions in the FASLG-Fas system.

What are the best experimental designs to study FASLG-induced apoptosis pathways?

Designing robust experiments to study FASLG-induced apoptosis requires consideration of multiple factors:

Cell model selection:

• Jurkat T cells: Widely used model for Fas-mediated apoptosis studies .

• Primary T lymphocytes: More physiologically relevant but technically challenging .

• HEK 293 cells expressing Fas: Useful for reconstitution experiments but require verification of pathway components .

Stimulation approaches:

• Recombinant FASLG proteins: Using soluble FASLG-Fc chimeras (6-36 ng/mL) in the presence of cross-linking enhancers .

• Antibody-mediated clustering: Anti-Fas antibodies can mimic FASLG stimulation.

• Membrane-bound FASLG: Co-culture with FASLG-expressing cells for more physiological stimulation.

Readout methods for apoptosis:

• Flow cytometry with Annexin V/PI staining

• Caspase activity assays

• Western blotting for cleaved PARP or caspases

• Live-cell imaging with fluorescent reporters

Controls and validation:

• Include blocking experiments with Fas-Fc (20-100 μg/mL) to confirm specificity .

• Use caspase inhibitors to verify the apoptotic mechanism.

• Compare wild-type and mutant Fas constructs (e.g., ΔDD or ΔPLAD mutations) .

These methodological considerations ensure reliable and interpretable results when studying FASLG-mediated apoptosis pathways.

How does post-transcriptional regulation affect FASLG expression in activated T cells?

FASLG expression is subject to complex post-transcriptional regulation, particularly in activated T cells:

AU-rich elements (AREs) and RNA-binding proteins:

• The FASLG 3′-untranslated region (UTR) contains two AU-rich elements (AREs) similar in sequence and structure to those in TNFα mRNA .

• HuR (ELAV-like RNA-binding protein) binds to these AREs both in vitro and ex vivo following T cell activation .

• This interaction stabilizes FASLG mRNA, increasing its half-life in activated T cells .

Experimental approaches to study post-transcriptional regulation:

• Immunoprecipitation followed by RT-PCR: This technique confirms the association between HuR and FASLG mRNA in activated Jurkat cells .

• Reporter gene assays: GFP reporter constructs fused to the FASLG 3′-UTR can be used to assess regulatory effects .

• HuR knockdown experiments: Silencing HuR prevents phorbol 12-myristate 13-acetate-induced expression of FASLG reporter constructs .

Physiological relevance:

• Post-transcriptional regulation provides an additional layer of control over FASLG expression, which is critical for proper immune function .

• Dysregulation of this mechanism could contribute to autoimmune disorders or immunodeficiency syndromes .

Understanding these regulatory mechanisms is essential for developing strategies to modulate FASLG expression in therapeutic applications.

What are the critical differences between membrane-bound and soluble forms of human FASLG?

The biological activities of membrane-bound versus soluble FASLG differ significantly, impacting experimental design and interpretation:

Structural and functional comparisons:

Key experimental considerations:

• Natural soluble FASLG has limited apoptotic activity compared to membrane-bound form .

• Engineered soluble FASLG with oligomerization domains (e.g., leucine zippers, Fc fusions) significantly enhances activity .

• Cross-linking with antibodies can further increase the apoptotic potential of soluble FASLG-Fc chimeras by 20-50 fold .

Applications in research:

• FasL-LZ (leucine zipper-fused FASLG) is valuable for receptor reorganization studies .

• FASLG-Fc chimeras are useful for inhibition studies, binding at 10-100 ng/ml in ELISA applications .

• Membrane-bound FASLG expression systems provide more physiologically relevant stimulation.

These differences must be carefully considered when designing experiments and interpreting results involving FASLG-mediated signaling.

How can I differentiate between specific and non-specific effects in FASLG-based experiments?

Distinguishing specific from non-specific effects is critical in FASLG research:

Validation strategies:

• Blocking experiments:

  • Use Fas-Fc fusion proteins (20-100 μg/ml) to specifically inhibit FASLG-Fas interactions .

  • Employ neutralizing antibodies against FASLG or Fas receptor.

  • The efficacy of blocking can be enhanced approximately 20-50 fold using cross-linking enhancers .

• Genetic approaches:

  • Compare responses in Fas-deficient vs. wild-type cells.

  • Use domain deletion mutants (e.g., ΔDD, ΔPLAD) or point mutations (e.g., A257D, E261K) in Fas receptor .

  • CRISPR-Cas9 knockout of pathway components can confirm specificity.

• Dose-response relationships:

  • Establish clear dose-response curves for FASLG effects (typically 6-36 ng/mL for recombinant proteins) .

  • Non-specific effects often lack the characteristic sigmoidal dose-response pattern.

• Pathway verification:

  • Monitor multiple steps in the apoptotic cascade (receptor clustering, FADD recruitment, caspase activation).

  • Use pathway inhibitors at different levels to confirm the involvement of specific signaling components.

• Controls for recombinant proteins:

  • Include irrelevant proteins with similar tags as negative controls.

  • Verify protein quality by SDS-PAGE and functional assays before use .

These approaches help ensure that observed effects are specifically attributable to FASLG-Fas interactions rather than experimental artifacts or off-target effects.

What are the potential applications of human FASLG in therapeutic development?

Human FASLG has several promising therapeutic applications under investigation:

Cancer immunotherapy:

• FASLG can directly induce apoptosis in Fas-expressing tumor cells.

• Engineered FASLG derivatives with enhanced stability and targeting could serve as novel cancer therapeutics .

• Combination approaches, such as with c-Met and Src inhibitors, show synergistic effects in inducing caspase-dependent apoptosis in certain cancer cells .

Autoimmune disease treatment:

• Modulating FASLG-Fas interactions could help restore immune tolerance in autoimmune conditions.

• Targeted delivery of FASLG to specific immune cell populations might reduce pathogenic immune responses while minimizing systemic effects .

Transplantation medicine:

• FASLG-based approaches could help induce tolerance to transplanted tissues.

• Local expression or delivery of FASLG might reduce rejection responses.

Diagnostic and prognostic applications:

• Soluble FASLG levels serve as biomarkers for disease activity in certain autoimmune conditions and cancers .

• Monitoring FASLG expression patterns in biopsies may provide prognostic information.

The translation of hFasLECD into an established agent in medicine is ongoing, with research focusing on improving stability, specificity, and delivery methods for these potential applications .

What are the latest techniques for studying FASLG-mediated signaling at the molecular level?

Cutting-edge techniques have significantly advanced our understanding of FASLG-mediated signaling:

Super-resolution microscopy approaches:

• Structured Illumination Microscopy (SIM) reveals Fas receptor reorganization following FASLG stimulation .

• Photoactivated Localization Microscopy (PALM) provides nanometer-scale resolution of receptor clustering dynamics .

• These techniques offer unprecedented insights into the spatial organization of signaling complexes.

Quantitative interaction analysis:

• Fluorescence Resonance Energy Transfer (FRET) with mCerulean3 and mVenus-tagged constructs enables real-time monitoring of protein-protein interactions in living cells .

• Flow cytometric FRET measurements provide population-level analysis of molecular interactions .

Systems biology approaches:

• Computational modeling of FASLG-Fas signaling networks integrates multiple experimental datasets.

• Proteomics and phosphoproteomics reveal the broader signaling networks activated by FASLG.

Gene editing technologies:

• CRISPR-Cas9 enables precise modification of FASLG or pathway components.

• Knock-in of fluorescent tags at endogenous loci allows visualization of native protein dynamics.

Single-cell analyses:

• Single-cell RNA-seq captures the heterogeneity in responses to FASLG stimulation.

• Mass cytometry (CyTOF) simultaneously measures multiple signaling events at the single-cell level.

These advanced techniques are revealing new insights into the complex dynamics and regulation of FASLG-mediated signaling pathways.

How do variations in experimental models affect FASLG research outcomes?

Experimental model selection significantly impacts FASLG research outcomes:

Cell line considerations:

Critical experimental variables:

• Activation state: FASLG responses differ dramatically between resting and activated lymphocytes .

• FasL formulation: Membrane-bound vs. soluble, degree of oligomerization, presence of cross-linking agents .

• Culture conditions: Serum factors can influence FASLG activity and stability .

• Cell density and health: Affect sensitivity to FASLG-induced apoptosis.

• Assay timing: FASLG-induced effects have distinct temporal dynamics.

Recommendations for robust research:

• Validate key findings across multiple model systems.

• Clearly report all experimental conditions and model characteristics.

• Consider both membrane-bound and soluble FASLG forms when appropriate.

• Include both short-term and long-term readouts for comprehensive understanding.

These considerations help ensure that research findings are robust and physiologically relevant, facilitating translation to clinical applications.