Recombinant Mouse Interleukin-4 (Il4), partial (Active)

What is the molecular structure of recombinant mouse IL-4?

Recombinant mouse IL-4 is a single, non-glycosylated polypeptide chain consisting of 121 amino acid residues. The typical amino acid sequence spans His23-Ser140, with an N-terminal Met addition when expressed in bacterial systems. The molecular mass ranges from 13.2 to 13.7 kDa as determined by SEC-MALS (Size Exclusion Chromatography-Multi-Angle Light Scattering) analysis, confirming that the protein exists primarily as a monomer in solution .

How is biological activity of recombinant mouse IL-4 measured?

The biological activity of recombinant mouse IL-4 is primarily measured through a proliferation assay using HT-2 mouse T cell lines. The effective dose for 50% maximal response (ED50) typically ranges between 0.2-1.5 ng/mL, corresponding to a specific activity of ≥5×10^6 U/mg . This standardized assay is calibrated against reference standards such as those provided by the WHO/National Institute for Biological Standards and Control (NIBSC code 91/656) .

For researchers conducting activity assays, it's important to note that biological response curves are typically sigmoidal, and accurate determination requires testing multiple concentrations spanning at least one log above and below the expected ED50. Results should be analyzed using appropriate curve-fitting software to determine precise activity metrics .

What are the recommended storage and reconstitution protocols?

Recombinant mouse IL-4 is typically supplied as a lyophilized product that should be stored at -20°C until reconstitution. Upon receipt, the product should be immediately stored at the recommended temperature to maintain stability and biological activity .

For reconstitution:

• Lyophilized mouse IL-4 should be reconstituted using deionized sterile-filtered water to a final concentration of 0.1–1.0 mg/mL in a minimal volume of 100 μL

• For preparations containing BSA as a carrier protein, reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• For carrier-free preparations, reconstitute at 100 μg/mL in sterile PBS

• Further dilutions should be prepared with buffer containing 0.1% bovine serum albumin (BSA) or human serum albumin (HSA) in phosphate-buffered saline

After reconstitution, it is recommended to aliquot the protein to avoid repeated freeze-thaw cycles, which can diminish biological activity. Reconstituted aliquots should be stored at –20°C or below for optimal stability .

How do splice variants of IL-4 differ in their functional activities?

While conventional IL-4 is encoded by four exons, an alternatively spliced isoform known as IL-4δ2 is encoded by exons 1, 3, and 4, omitting exon 2. Experimental studies comparing human IL-4 isoforms in mouse models have revealed significant functional differences. Both isoforms cause similar pulmonary infiltration of T and B lymphocytes when delivered to mouse lungs using replication-deficient adenovirus-mediated gene delivery, but notably, they do not induce eosinophil infiltration .

The most striking differences between these isoforms appear in their modulation of the cytokine milieu. IL-4δ2 induces higher levels of pro-inflammatory cytokines (TNF-α, IL-1, MCP-1) and Th1 cytokines (IL-12, IFN-γ) compared to conventional IL-4. This indicates that IL-4δ2 may have a more pronounced pro-inflammatory effect than conventional IL-4, suggesting potentially distinct signaling mechanisms or receptor engagement patterns .

These findings have important implications for experimental design, as researchers should carefully consider which IL-4 isoform is most appropriate for their specific research questions, especially in studies examining inflammatory responses or Th1/Th2 balance.

What are the critical factors affecting IL-4 receptor signaling?

IL-4 receptor signaling involves complex interactions between IL-4 and its receptors, with several critical molecular determinants. The human IL-4 receptor contains a "gene regulation domain" between amino acids 558-657 that is essential for signaling. Within this domain, three conserved tyrosine residues (Y2, Y3, and Y4) play crucial roles in signal transduction .

For researchers studying IL-4 signaling:

• Consider potential differences in signaling mechanisms between mouse and human IL-4 systems

• Evaluate receptor expression levels when interpreting signaling experiments, as high receptor density may compensate for partial signaling defects

• Remember that extended domain structure plays an essential role in the recognition and function of juxtatyrosine sequences in IL-4R signaling

What are the cross-species considerations when using mouse IL-4 in experimental systems?

Cross-species activity of IL-4 presents both challenges and opportunities for researchers. Structural analysis indicates that key residues in human IL-4 that define its binding to human IL-4Rα (Glu-9 and Arg-88), as well as minor determinants (Arg-53 and Arg-85), are all present in mouse IL-4. Direct binding experiments using BIAcore systems have confirmed that glycosylated mouse IL-4 can bind to human IL-4 receptor α (IL-4Rα) .

Multiple studies have demonstrated that human IL-4 can be active in mice or rats in vivo, functioning similarly to mouse IL-4. This cross-species activity enables at least limited testing of anti-human IL-4 therapies in mouse models, creating opportunities for translational research .

• Potentially higher doses may be required for cross-species applications

• Validation of receptor binding and downstream signaling is essential

• Comparisons with species-matched controls should be included whenever possible to assess relative activity

What are the optimal expression systems for producing recombinant mouse IL-4?

E. coli Expression Systems:

• The BL21(DE3)-CodonPlus E. coli strain with pET-21b (+) vector has been successfully used to express mouse IL-4, though the protein is often produced as an insoluble molecule requiring refolding

• After induction with IPTG, the expressed protein should be analyzed by SDS-PAGE to confirm correct size (typically 13-17.5 kDa depending on the construct)

• If the protein is expressed in inclusion bodies, solubilization with guanidine hydrochloride and dithiothreitol followed by refolding and purification by chromatography may be necessary

Purification Considerations:

• Western blot analysis using anti-IL-4 specific antibodies should be performed to confirm the identity of the purified protein

• The purity should exceed 97% as determined by SDS-PAGE analysis

• Endotoxin levels should be measured and maintained below 1.0 EU/μg to avoid confounding experimental results

For researchers requiring glycosylated IL-4, mammalian expression systems may be preferable, though they typically yield lower amounts of protein compared to bacterial systems.

How should IL-4 bioactivity be validated in experimental systems?

Validating the bioactivity of recombinant IL-4 is critical for experimental reproducibility. A multi-faceted approach is recommended:

Proliferation Assays:

• HT-2 mouse T cell proliferation assay remains the gold standard, with expected ED50 values between 0.2-1.5 ng/mL

• Cell viability should be measured using an appropriate method (MTT, XTT, or alamarBlue assays)

• A dose-response curve using at least 8 concentration points spanning 0.01-10 ng/mL should be established

Receptor Binding Confirmation:

• Surface plasmon resonance (e.g., BIAcore) to measure direct binding kinetics to IL-4Rα

• Flow cytometry to assess binding to receptor-expressing cells

Downstream Signaling Validation:

• Western blot analysis of STAT6 phosphorylation

• Reporter gene assays for IL-4-responsive elements

• qPCR measurement of IL-4-induced genes (e.g., CD23)

Activity Comparison Table:

What are the critical considerations for designing IL-4 treatment experiments?

When designing experiments involving recombinant mouse IL-4 treatment, researchers should consider several factors to ensure robust and reproducible results:

Dosage Determination:

• Conduct preliminary dose-response studies covering at least 3 logs (e.g., 0.1-100 ng/mL)

• For in vivo studies, consider both local concentration at target tissues and systemic distribution

• Account for potential differences in potency between different preparations of IL-4

Timing and Duration:

• Acute vs. chronic exposure should be determined based on research objectives

• For signaling studies, early time points (5-60 minutes) are critical for capturing phosphorylation events

• For gene expression changes, later time points (2-24 hours) may be more appropriate

• For phenotypic changes (e.g., T cell differentiation), extended exposure (3-7 days) is typically required

Delivery Methods:

• In vitro: Direct addition to culture medium, with consideration of protein stability over time

• In vivo: Consider adenovirus-mediated gene delivery for sustained expression, as demonstrated for IL-4 isoforms in lung studies

• For localized delivery, options include osmotic pumps or matrix-embedded slow-release formulations

Control Conditions:

• Vehicle control (buffer with matching carrier protein concentration)

• Heat-inactivated IL-4 as a negative control

• Species-matched positive controls for cross-species experiments

• Consider including other cytokines (e.g., IL-13) to distinguish IL-4-specific effects from shared receptor signaling

How can variable or contradictory results with IL-4 treatments be resolved?

Researchers occasionally encounter variable or contradictory results when using recombinant IL-4 in experiments. Several approaches can help resolve these issues:

Protein Quality Assessment:

• Verify activity using standardized bioassays (HT-2 proliferation)

• Check for protein degradation by SDS-PAGE and Western blot

• Measure endotoxin levels, as contamination can confound immunological experiments

Receptor Expression Analysis:

• Quantify IL-4Rα expression levels on target cells, as receptor density impacts signaling sensitivity

• Consider analyzing common IL-4R polymorphisms that might affect responsiveness

• Remember that high receptor expression can compensate for partial signaling defects in IL-4R mutants

Experimental Conditions Audit:

• Document exact protocol parameters including cell density, media composition, and timing

• Assess the impact of serum components, which may contain IL-4 binding proteins

• Control for inadvertent activation of cells during processing, which can alter cytokine responsiveness

Reconciliation Strategies:

• Directly compare IL-4 preparations using side-by-side assays

• Use multiple readouts (e.g., STAT6 phosphorylation, gene expression, and functional responses)

• Consider the presence of inhibitory factors or receptor antagonists in your experimental system

What are common pitfalls in IL-4 functional studies and how can they be avoided?

Several common pitfalls can compromise IL-4 functional studies. Awareness and preventive measures include:

Storage and Handling Issues:

• Avoid repeated freeze-thaw cycles that diminish biological activity

• Prepare single-use aliquots after reconstitution

• Use a manual defrost freezer for storage to maintain consistent temperature

Reconstitution Errors:

• Follow manufacturer's recommendations for reconstitution buffer and concentration

• Include carrier protein (0.1% BSA or HSA) for dilutions to prevent loss through adsorption to plastics

• Allow complete solubilization before use (gentle swirling rather than vortexing)

Experimental Design Flaws:

• Inadequate concentration range in dose-response studies

• Insufficient time points to capture transient signaling events

• Lack of appropriate positive and negative controls

• Failure to account for endogenous IL-4 production in mixed cell populations

Data Interpretation Challenges:

• Over-interpretation of in vitro findings without in vivo validation

• Failure to consider IL-4 isoform-specific effects, as conventional IL-4 and IL-4δ2 may have distinct functions

• Not accounting for differences between human and mouse IL-4 systems when translating findings across species

• Overlooking the importance of juxtatyrosine residues and extended domain structures in IL-4R signaling

How can researchers differentiate between direct and indirect effects of IL-4 in complex experimental systems?

Distinguishing direct IL-4 effects from indirect consequences in complex systems presents a significant challenge. Methodological approaches to address this include:

Receptor Blocking Studies:

• Use neutralizing antibodies against IL-4 or IL-4Rα

• Apply soluble IL-4 receptors to competitively inhibit IL-4 binding

• Compare results with IL-4Rα knockout or knockdown models

Cell-Specific Response Analysis:

• Isolate individual cell populations before and after IL-4 treatment

• Use cell type-specific markers in flow cytometry or immunohistochemistry

• Employ single-cell RNA sequencing to identify direct responders

• Consider conditional IL-4Rα knockout models targeting specific cell populations

Timing Analysis:

• Establish a detailed time course to distinguish primary (rapid) from secondary (delayed) responses

• Compare kinetics of different outcome measures to establish likely causal relationships

• Use transcription or translation inhibitors to block secondary effects

Pathway Delineation:

• Target specific components of the IL-4 signaling cascade (e.g., JAK1, STAT6) to confirm mechanistic involvement

• Compare responses to IL-13, which shares the Type II IL-4 receptor but may elicit distinct outcomes

• Consider the functional differences between IL-4 isoforms, as IL-4δ2 induces different cytokine profiles compared to conventional IL-4

What are the key considerations when comparing results across different recombinant IL-4 preparations?

When comparing experimental results obtained using different recombinant IL-4 preparations, researchers should consider several variables that can impact outcomes:

Protein Characteristics:

• Sequence variations: Confirm identical amino acid sequences (His23-Ser140 with N-terminal Met is standard for E. coli-derived mouse IL-4)

• Post-translational modifications: E. coli-derived IL-4 lacks glycosylation, which may affect activity compared to mammalian-expressed protein

• Purity: Standardize to >97% purity as determined by SDS-PAGE analysis

• Endotoxin levels: Ensure comparable and low endotoxin content (<1.0 EU/μg)

Activity Standardization:

• Normalize treatments based on specific activity rather than mass concentration

• Consider calibrating against reference standards such as WHO/NIBSC preparations (code 91/656)

• Perform side-by-side bioactivity assays when directly comparing different preparations

• Document ED50 values (expected range: 0.2-1.5 ng/mL for HT-2 proliferation)

Experimental Design Harmonization:

• Standardize carrier protein concentrations across preparations

• Use consistent reconstitution and storage protocols

• Apply identical experimental conditions (cell density, media composition, incubation times)

• Include internal controls to normalize between experiments

How might emerging research impact our understanding of IL-4 biology and applications?

Recent and ongoing research continues to expand our understanding of IL-4 biology in ways that may impact experimental approaches:

Isoform-Specific Functions:
Research on splice variants such as IL-4δ2 reveals distinct functional profiles compared to conventional IL-4. IL-4δ2 appears to induce stronger pro-inflammatory and Th1 responses, suggesting potential specialized roles in immune regulation. Future studies may uncover additional splice variants with unique functions .

Cross-Species Applications:
The discovery that human IL-4 can be functionally active in mice opens new possibilities for translational research. This cross-species activity enables limited pre-clinical testing of human IL-4-targeting therapies in animal models, potentially accelerating therapeutic development .

Receptor Signaling Complexity:
The importance of extended domain structure and juxtatyrosine sequences in IL-4R signaling indicates more complex receptor-cytokine interactions than previously appreciated. Future research may reveal additional structural determinants of signaling specificity and strength .

Emerging Technologies:
New methodologies including CRISPR-based gene editing, single-cell analysis, and advanced structural biology techniques promise to further deepen our understanding of IL-4 biology. These approaches may reveal novel regulatory mechanisms, interaction partners, and therapeutic targets related to IL-4 signaling.

As research progresses, experimental design should adapt to incorporate these emerging insights, ensuring that IL-4 studies remain at the cutting edge of immunological research.