IL5 Mouse, Sf9

What is IL5 Mouse, Sf9 and what are its main biological functions?

IL5 Mouse, Sf9 refers to murine Interleukin 5 produced in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. It is a cytokine that functions as a growth and differentiation factor for both B cells and eosinophils. This protein plays a critical role as a main regulator of eosinopoiesis, eosinophil maturation and activation. The elevated production of IL5 has been linked to asthma and hypereosinophilic syndromes .

From a functional perspective, IL5 is biologically active in inducing cell-mediated immunity against parasitic infections and certain tumors. In mouse models specifically, IL5 demonstrates activity on B cells, a function that is less pronounced in human systems . IL5 promotes the production and mobilization of eosinophils and CD34+ progenitors from the bone marrow .

How does mouse IL5 differ from human IL5 in terms of biological activity?

Mouse and human IL5 exhibit significant differences in their biological activities despite sharing approximately 70% sequence similarity. Key differences include:

• Species-specific activity: While mouse and human IL5 are equally active in human cell assays, human IL5 is approximately 100-fold less active than murine IL5 in mouse cell assays .

• B cell activity: Mouse IL5 demonstrates pronounced activity on B cells, a function that is less evident with human IL5 .

• Receptor binding: Structure-function analyses using mouse/human chimeras have identified that the C-terminus region is particularly important for biological activity. Changing only eight residues in this region of human IL5 to those of mouse IL5 results in biological activity comparable to mouse IL5 in mouse systems .

• Binding competition assays have demonstrated that these C-terminal residues likely interact directly with the receptor, explaining the species-specific differences in activity .

These differences are critical considerations when designing experiments and interpreting results across different model systems.

What expression systems are most effective for producing functional mouse IL5?

Multiple expression systems have been employed for producing functional mouse IL5, each with distinct advantages depending on research requirements:

• Sf9 Baculovirus System: This insect cell expression system provides high yields of properly folded, glycosylated IL5. The baculovirus expression vector system allows for efficient production of recombinant proteins with post-translational modifications similar to mammalian systems . This system is particularly valuable for structural studies and functional assays.

• CHO Cell Expression: Chinese Hamster Ovary cells produce highly active IL5 with mammalian glycosylation patterns. CHO-expressed IL5 demonstrates high specific activity (>2×10^6 units/mg) with ED50 values <0.5 ng/ml in TF-1 cell proliferation assays . This system may be preferred when mammalian glycosylation is essential for the experimental design.

• Bacterial Expression Systems: Though not detailed in the provided search results, E. coli systems are sometimes used for non-glycosylated variants, particularly for structural studies.

The choice depends on the specific research question, with Sf9 and CHO systems generally preferred for functional studies due to their ability to perform proper folding and post-translational modifications.

What purification strategies yield the highest activity for IL5 Mouse from Sf9 cells?

Effective purification of IL5 Mouse from Sf9 cells typically follows a multi-step approach:

• Initial Clarification: The supernatant from Sf9 culture is harvested and filtered to remove cellular debris.

• Affinity Chromatography: For His-tagged constructs (as indicated by the C-terminal HHHHHH sequence in the provided sequence), immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins provides efficient initial purification .

• Further Purification: Size exclusion chromatography (SEC) and/or ion exchange chromatography are often employed as secondary purification steps to achieve higher purity.

The final purified product typically achieves >90% purity as determined by SDS-PAGE . The purification process should be optimized to maintain the dimeric structure of IL5, which is essential for its biological activity.

To preserve activity during purification, it is advisable to include stabilizing agents such as glycerol (typically 10%) in buffer formulations .

What quality control methods should be employed to verify IL5 functionality?

Multiple quality control methods should be implemented to verify the functionality of purified IL5 Mouse:

• Biological Activity Assays: Cell proliferation assays using TF-1 human erythroleukemic cells are the gold standard for determining IL5 activity. Functional IL5 Mouse from Sf9 should demonstrate an ED50 value of approximately 1 ng/ml . For CHO-expressed IL5, ED50 values of <0.5 ng/ml have been reported .

• Structural Verification:

  • SDS-PAGE analysis to confirm purity (>90%)

  • Western blot using specific anti-IL5 antibodies

  • ELISA using different monoclonal antibodies (mAbs) such as H30 (non-neutralizing) or SA5 (neutralizing)

• Receptor Binding Assays: Solid-phase binding assays using soluble IL5 receptor-alpha (IL5Rα) fusion proteins can verify receptor interaction capability .

• Species-Specific Activity: Testing in both human and mouse cell systems to confirm the expected species-specific activity profiles .

What are the optimal storage conditions for maintaining IL5 Mouse, Sf9 activity?

The optimal storage conditions for IL5 Mouse, Sf9 depend on the intended duration of storage:

• Short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this timeframe .

• Long-term storage: Store frozen at -20°C. For very long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) to prevent activity loss .

• Working solution: The IL5 solution (typically 0.25mg/ml) should be maintained in phosphate-buffered saline (pH 7.4) with 10% glycerol as a stabilizing agent .

Critical considerations include:

• Avoiding multiple freeze-thaw cycles, which can significantly decrease biological activity

• Aliquoting the stock solution before freezing to minimize freeze-thaw cycles

• Ensuring sterile handling conditions to prevent microbial contamination

How can researchers assess potential activity loss during storage?

To assess potential activity loss during storage, researchers should:

• Establish a baseline: Measure the initial biological activity of freshly prepared IL5 using the TF-1 cell proliferation assay, determining the ED50 value (approximately 1 ng/ml for fully active protein) .

• Periodic testing: At defined intervals, test aliquots of the stored IL5 using the same standardized assay.

• Comparative analysis: Calculate the percentage of activity retention by comparing current ED50 values to the baseline. A significant increase in ED50 (requiring more protein to achieve the same effect) indicates activity loss.

• Structural integrity assessment: In parallel with activity assays, researchers can perform SDS-PAGE and/or ELISA using specific antibodies to detect potential structural changes or degradation .

• Control inclusion: Always include a reference standard of known activity (commercially available or well-characterized internal standard) in each assay to normalize results and account for inter-assay variation.

A systematic approach to monitoring stability can help establish appropriate storage periods and conditions for specific research applications.

What cell-based assays are most reliable for measuring IL5 Mouse activity?

Several cell-based assays have been validated for measuring IL5 Mouse activity, each with specific advantages:

• TF-1 Human Erythroleukemic Cell Proliferation: This is the most widely used and standardized assay for IL5 activity. TF-1 cells respond to IL5 with a dose-dependent proliferation that can be quantitatively measured. The typical ED50 for IL5 Mouse in this assay is approximately 1 ng/ml .

• FDCP1-CA1 Cell Proliferation: This cell line is derived from FDC-P1 cells by introducing human IL5Rα cDNA, making it useful for assessing receptor-specific activities. It provides a complementary system to verify activity measurements .

• TF1-hIL5Rα Cell Assay: This human erythroleukemia cell line derivative has been engineered to express human IL5Rα, providing a standardized system for comparing activity across different IL5 preparations .

• Primary Mouse B Cell and Eosinophil Assays: For more physiologically relevant assessments, especially when studying species-specific activities, primary cell assays using mouse bone marrow-derived eosinophils or B13 pro-B cells can be employed .

The choice of assay depends on the specific research question, with TF-1 proliferation being the most standardized for general activity measurements.

How can researchers effectively perform structure-function studies on IL5 Mouse?

Structure-function studies on IL5 Mouse can be effectively performed using several complementary approaches:

• Site-Directed Mutagenesis: Introducing specific mutations into the IL5 sequence allows for the identification of critical residues involved in receptor binding and activation. Complete or partial alanine scanning (replacing residues with alanine) of charged residues has been particularly informative .

• Mouse/Human Chimeras: Creating hybrid molecules by swapping segments between mouse and human IL5 enables the identification of regions responsible for species-specific activities. This approach identified the C-terminus as critical for species specificity .

• Receptor Binding Assays: Solid-phase binding assays using soluble IL5Rα-IgG fusion proteins can quantitatively measure how mutations affect receptor binding. This can be performed as a competition assay with labeled IL5 .

• Functional Bioassays: Testing mutants in both proliferation assays and receptor binding assays allows researchers to distinguish between mutations affecting receptor binding versus those affecting receptor activation .

• Expression Verification: For all mutants, expression should be verified using metabolic labeling (e.g., with [35S]methionine) followed by immunoprecipitation with anti-IL5 antibodies and SDS-PAGE analysis .

This multi-faceted approach can provide comprehensive insights into structure-function relationships.

What are the critical considerations when comparing IL5 produced in different expression systems?

When comparing IL5 Mouse produced in different expression systems (e.g., Sf9 versus CHO cells), researchers should consider the following critical factors:

• Glycosylation Patterns: Different expression systems produce distinct glycosylation profiles, which can affect protein stability, half-life, and receptor interactions. Insect cells (Sf9) produce simpler glycosylation patterns compared to mammalian cells (CHO) .

• Specific Activity: Quantitative comparison of specific activity (units/mg) is essential. CHO-expressed IL5 has been reported with specific activity >2×10^6 units/mg and ED50 <0.5 ng/ml in TF-1 assays, while Sf9-expressed IL5 typically shows ED50 of approximately 1 ng/ml .

• Structural Integrity: The dimeric structure of IL5 is essential for its activity. Verify that both expression systems maintain proper protein folding and dimerization.

• Endotoxin Levels: Different expression systems may introduce varying levels of endotoxin contamination, which can confound biological assays, particularly those involving immune cells.

• Experimental Context: Consider whether the specific research application requires mammalian glycosylation patterns (use CHO) or if higher yields are the priority (Sf9 may provide advantages).

• Standardization: Include reference standards in all comparative studies to normalize results and account for inter-assay variation.

A systematic comparison across multiple parameters will provide the most reliable basis for selecting the appropriate expression system for specific research applications.

How can IL5 receptor antagonists be designed based on structure-function knowledge?

Designing IL5 receptor antagonists requires a sophisticated understanding of structure-function relationships. The following methodological approach has proven effective:

• Target Critical Binding Regions: Focus on residues identified through mutagenesis studies as essential for receptor binding. Key regions include β-strand 2 (residues E89, R91), the loop between β-strand 1 and helix B (H38, K39, H41), and the end of helix D (T109, E110, W111, I112) .

• Create Binding Site Mutants: Develop mutations that maintain receptor binding but disrupt receptor activation. This approach was used successfully in developing antagonistic IL5 muteins .

• Exploit Species Differences: Utilize knowledge from human/mouse chimera studies, particularly focusing on the C-terminal region which has been shown to interact directly with the receptor .

• Verify Antagonist Properties:

  • Confirm binding to IL5Rα using solid-phase binding assays

  • Demonstrate lack of proliferative activity in IL5-dependent cell lines

  • Show competitive inhibition of wild-type IL5 activity

  • Assess specificity by testing effects on related cytokine pathways

• Structural Optimization: Once initial antagonists are identified, perform further structural refinements to improve binding affinity, stability, and pharmacokinetic properties.

This systematic approach has led to the development of antagonistic IL5 muteins with potential therapeutic applications in asthma and hypereosinophilic disorders .

What are common challenges in IL5 functional assays and how can they be addressed?

Several challenges commonly arise in IL5 functional assays, along with effective solutions:

• Activity Variability:

  • Challenge: Inconsistent ED50 values between assays

  • Solution: Include internal standards in each assay; normalize results against reference preparations; ensure consistent cell passage numbers and assay conditions

• Species-Specific Effects:

  • Challenge: Different responses in human versus mouse systems

  • Solution: Always interpret results in the context of the specific species being studied; when comparing across species, use chimeric constructs as reference points

• Receptor Expression Levels:

  • Challenge: Variable receptor expression on cell lines affecting sensitivity

  • Solution: Regularly verify receptor expression by flow cytometry; consider using engineered cell lines with stable, defined receptor expression

• Protein Stability:

  • Challenge: Activity loss during storage or experimental manipulation

  • Solution: Add carrier proteins (0.1% HSA or BSA) for stability; avoid freeze-thaw cycles; maintain glycerol (10%) in storage buffers

• Non-Specific Binding in Receptor Assays:

  • Challenge: Background signal in binding assays

  • Solution: Optimize blocking conditions; use competition assays with labeled IL5 rather than direct binding measurement

• Distinguishing Receptor Binding from Activation:

  • Challenge: Separating these two aspects of IL5 function

  • Solution: Employ both binding assays (solid-phase with soluble receptor) and functional assays (cell proliferation); compare results to identify mutations affecting one function but not the other

Addressing these challenges through methodological refinements increases assay reliability and facilitates meaningful interpretation of results.

How do post-translational modifications affect IL5 Mouse functionality?

Post-translational modifications (PTMs) significantly impact IL5 Mouse functionality across several dimensions:

• Glycosylation:

  • IL5 is a glycosylated protein, and the glycosylation pattern affects stability, half-life, and receptor interaction kinetics

  • Different expression systems (Sf9 versus CHO) produce distinct glycosylation profiles, potentially affecting functional properties

  • Glycosylation may protect certain regions of the protein from proteolytic degradation

• Disulfide Bonding:

  • Proper disulfide bond formation is critical for maintaining the tertiary structure of IL5

  • Expression systems with appropriate oxidative environments are essential for functional protein production

• Proteolytic Processing:

  • The mature IL5 protein corresponds to amino acids 21-133 of the precursor, requiring proper proteolytic processing

  • Improper processing can affect N-terminal structure and function

• Dimerization:

  • IL5 functions as a dimer, and conditions promoting appropriate dimerization are essential for activity

  • Some assays, such as certain ELISAs using the SA5 monoclonal antibody, take advantage of the dimeric structure for detection

When designing experiments:

• Consider the impact of expression system choice on PTMs

• Verify the structural integrity of the protein through multiple methods

• Assess functionality through both binding and cell-based assays

• For critical applications, characterize the PTM profile using mass spectrometry or similar techniques

Understanding and controlling these PTM effects is essential for reproducible research outcomes and meaningful comparisons between different IL5 preparations.

How does IL5 Mouse signaling integrate with other cytokine networks?

IL5 Mouse functions within a complex cytokine network, with several important interactions:

• Genomic Clustering and Co-regulation: IL5 forms a cytokine gene cluster on chromosome 5 together with IL4, IL13, and CSF2. These cytokines are regulated coordinately by long-range regulatory elements spread over 120 kilobases on chromosome 5q31, suggesting functional relationships in immune responses .

• Shared Receptor Components: The IL5 receptor beta subunit is shared with the receptors for IL3 and CSF2/GM-CSF, indicating overlapping signaling pathways and potential functional redundancy in some contexts .

• Cooperative Effects: In research contexts, IL5 often works synergistically with:

  • IL3 and GM-CSF in promoting eosinophil development

  • IL4 and IL13 in allergic responses and asthma pathogenesis

  • IL2 in certain T cell and B cell activation scenarios

• Differential Species Effects: The broader cytokine network interactions may differ between mouse and human systems, explaining some of the species-specific activities observed with IL5 .

Understanding these network interactions is essential when:

• Designing experiments to isolate IL5-specific effects

• Interpreting results in complex biological systems

• Developing targeted interventions for conditions like asthma or hypereosinophilic syndromes

• Translating findings from mouse models to human applications

What quantitative methods provide the most accurate assessment of IL5-receptor interactions?

Several quantitative methods provide robust assessment of IL5-receptor interactions:

• Solid-Phase Competition Binding Assays:

  • Methodology: Plates are coated with anti-hIgG and loaded with soluble hIL5Rα-hIgG3 fusion protein. Competition between labeled IL5 and test samples provides quantitative binding data.

  • Advantage: This approach is approximately 5-fold more accurate than bioassays for quantifying binding interactions .

• Surface Plasmon Resonance (SPR):

  • While not explicitly mentioned in the search results, SPR allows real-time measurement of binding kinetics (kon and koff) between IL5 and its receptor.

  • Provides association and dissociation constants that characterize the binding interaction.

• Radioligand Binding Assays:

  • Using 125I-labeled IL5 in competition assays allows precise quantification of binding affinities.

  • This approach has been used successfully with mouse/human chimeras to identify regions critical for receptor interaction .

• Cell-Based Assays with Dose-Response Analysis:

  • Proliferation assays using IL5-dependent cell lines (FDCP1-CA1, TF1-hIL5Rα) with detailed dose-response curves.

  • ED50 values from these assays provide functional measures of receptor interaction .

When conducting these analyses, researchers should:

What are the key experimental design considerations when using IL5 Mouse, Sf9 in complex immunological studies?

When incorporating IL5 Mouse, Sf9 into complex immunological studies, researchers should consider several critical experimental design factors:

• Species Compatibility:

  • Mouse IL5 exhibits distinct species-specific activity patterns; it is fully active in both mouse and human systems, unlike human IL5 which has reduced activity in mouse systems .

  • This becomes particularly important in experiments using cells or tissues from different species.

• Concentration Standardization:

  • Establish dose-response relationships in preliminary experiments.

  • The ED50 of approximately 1 ng/ml in TF-1 proliferation assays serves as a reference point .

  • For comparative studies with different IL5 preparations, standardize based on biological activity rather than protein concentration alone.

• Receptor Expression Context:

  • The IL5 receptor consists of specific α chains and shared β chains. The cellular context and relative expression levels of these components can significantly affect responses .

  • Consider characterizing receptor expression in the experimental system.

• Functional Readouts:

  • Select appropriate readouts based on the cell type and expected response:

    • For eosinophils: differentiation, activation, survival

    • For B cells (in mouse systems): proliferation, antibody production

    • For broader studies: cytokine production, gene expression changes

• Controls and Validation:

  • Include both positive controls (known IL5-responsive systems) and negative controls (receptor-blocking antibodies or cells lacking IL5 receptors).

  • For critical findings, validate with independent approaches (e.g., genetic knockdown/knockout of receptors).

• Storage and Handling During Experiments:

  • Maintain protein stability throughout the experiment by avoiding repeated freeze-thaw cycles.

  • For long-term studies, prepare single-use aliquots with carrier protein (0.1% HSA or BSA) .

• Integration with Other Cytokines:

  • Consider the cooperative or antagonistic effects with related cytokines (IL3, GM-CSF, IL4, IL13).

  • In some cases, controlling for or blocking these related pathways may be necessary to isolate IL5-specific effects.

Careful attention to these experimental design considerations ensures robust, reproducible findings in complex immunological studies using IL5 Mouse, Sf9.