Recombinant Rhesus Macaque Interleukin-13 protein (IL13) (Active)

What is the structural composition of Recombinant Rhesus Macaque IL13?

Recombinant Rhesus Macaque IL13 is a protein that spans amino acids 19-132 of the full IL13 sequence. The protein has a molecular weight of approximately 12.6-14.3 kDa, with an accurate measured mass of 14 kDa in most preparations. The isoelectric point of the protein is 9.5, indicating its basic nature. The amino acid sequence includes specific regions that are critical for receptor binding and biological activity, with the full sequence comprising residues that form the functional domain of the protein .

When analyzing the protein structure through SDS-PAGE, researchers should expect to observe a single band at approximately 12.6-14.3 kDa, reflecting the monomeric form of the protein. The variation in molecular weight estimates between different sources likely reflects differences in expression constructs, with some versions including additional tags or fusion partners that affect mobility on gels .

How does Rhesus Macaque IL13 compare to human IL13 in research applications?

Rhesus Macaque IL13 shares significant homology with human IL13, making it a valuable research tool for translational studies. The protein functions as an immunoregulatory cytokine predominantly expressed by activated Th2 cells. In Rhesus macaques, IL13 plays a crucial role in regulating type 2 immunity, similar to its function in humans. The protein is associated with Th2 skewed responses and linked to eosinophil levels in the lungs of challenged animals .

Research has identified an IL13+GATA3+ Th2 subset in Rhesus macaques that expresses eicosanoid pathway enzymes, accompanied by IL1RL1+GATA3+ regulatory T cells and a minor proportion of IgE+ plasma cells. This demonstrates a tightly regulated type 2 immunity in these animals that parallels human immune regulation, making macaque models particularly valuable for studying allergic and inflammatory conditions .

What expression systems are used for producing Recombinant Rhesus Macaque IL13?

Recombinant Rhesus Macaque IL13 is primarily produced using prokaryotic expression systems, specifically Escherichia coli. The production process involves co-cloning the target gene encoding amino acids 19-132 of IL13 into an expression vector. This vector is then introduced into E. coli cells, which are cultured under optimized conditions for protein expression. Following cell culture and induction, the cells undergo lysis, and the recombinant protein is purified using affinity chromatography techniques .

What quality control parameters are essential for validating Recombinant Rhesus Macaque IL13?

Multiple quality control parameters should be assessed when validating Recombinant Rhesus Macaque IL13 for research applications:

• Purity assessment: High-quality preparations should demonstrate >90-98% purity as determined by SDS-PAGE analysis. This ensures minimal contamination with bacterial proteins or truncated forms of the target protein .

• Endotoxin testing: Endotoxin levels should be <1.0 EU/μg as determined by the Limulus Amebocyte Lysate (LAL) method. This is critical for preventing non-specific immune activation in cell culture or in vivo experiments .

• Biological activity: Functional testing using cell proliferation assays with human TF-1 cells should demonstrate an ED50 of less than 5 ng/ml, corresponding to a specific activity of >2.0×10^5 IU/mg. This confirms that the recombinant protein maintains its functional characteristics .

• Thermal stability: Accelerated thermal degradation testing (e.g., incubation at 37°C for 48 hours) should show minimal degradation and no precipitation, with loss rates less than 5% under appropriate storage conditions .

How can Recombinant Rhesus Macaque IL13 be utilized in asthma and allergy research models?

Recombinant Rhesus Macaque IL13 serves as an important tool in asthma and allergy research, particularly in studies using non-human primate models. When designing experiments with IL13 in asthma models, researchers should consider several methodological approaches:

• Direct administration studies: Recombinant IL13 can be administered to examine its effects on airway hyperresponsiveness, mucus production, and eosinophil recruitment. Typically, dosages range from 1-10 μg/kg based on similar cytokine studies, delivered via aerosol, intratracheal, or systemic routes depending on the experimental question .

• Antagonist efficacy testing: The protein serves as a positive control or target antigen when evaluating anti-IL13 therapeutic approaches. Research has shown that anti-IL13 monoclonal antibodies can be well-tolerated in both normal and allergic asthmatic macaques, providing important safety and pharmacodynamic data for therapeutic development .

• Biomarker identification: Studies utilizing IL13 have identified that serum eotaxin concentrations may serve as a useful early in vivo marker for evaluating IL13 inhibition in asthma. In macaque models challenged with Ascaris suum antigen, anti-IL13 antibody treatment produced a significant reduction in BAL and serum eotaxin concentrations at 6 hours post-challenge (p < 0.05) .

What methodological approaches can be used to study IL13-mediated immune responses in Rhesus macaque models?

Several methodological approaches can be employed to study IL13-mediated immune responses in Rhesus macaque models:

• Allergen challenge models: Ascaris suum antigen challenge has been established as an effective method to induce allergic responses in Rhesus macaques. When implementing this model, researchers should select macaques that demonstrate a positive bronchoconstrictor response to inhaled allergen. The antigen is typically administered via aerosol inhalation using intermittent positive pressure breathing with a ventilator and in-line ultrasonic nebulizer. This approach allows for the assessment of IL13's role in the asthmatic response .

• Cytokine profiling: To comprehensively assess the role of IL13 in immune responses, researchers should measure multiple cytokines simultaneously. This includes evaluation of IL13 in conjunction with other Th2 cytokines (IL4, IL5), as well as eotaxin levels in both serum and bronchoalveolar lavage (BAL) fluid. Time-course analyses (e.g., 6h, 24h, 48h post-challenge) provide insights into the kinetics of the immune response .

• Cell subset analysis: Flow cytometric identification of IL13+GATA3+ Th2 cells provides valuable information about the cellular sources and targets of IL13. Research has identified specific Th2 subsets in Rhesus macaques that express eicosanoid pathway enzymes, which should be considered when investigating IL13-mediated inflammation .

What are the optimal storage and handling protocols for maintaining IL13 stability?

To ensure optimal stability and activity of Recombinant Rhesus Macaque IL13, researchers should follow these storage and handling protocols:

• Reconstitution: The lyophilized protein should be reconstituted in 10mM PBS (pH 7.4) to a concentration of 0.1-1.0 mg/mL. It's important to avoid vortexing during reconstitution to prevent protein denaturation .

• Short-term storage: For experiments requiring use within one month, the reconstituted protein can be stored at 2-8°C .

• Long-term storage: For extended storage periods, the protein should be aliquoted to minimize freeze-thaw cycles and stored at -80°C. Under these conditions, the protein remains stable for up to 12 months .

• Avoiding degradation: Repeated freeze/thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of biological activity. The thermal stability of properly stored protein should show less than 5% loss of activity within the expiration date .

• Working solution preparation: When preparing working dilutions for bioassays, the protein should be diluted in appropriate cell culture medium containing a carrier protein (e.g., 0.1% BSA) to prevent adsorption to plasticware and maintain stability .

How can researchers effectively measure IL13 activity in experimental systems?

Effective measurement of IL13 activity in experimental systems requires multiple complementary approaches:

• Cell proliferation assays: The standard bioactivity assay for IL13 utilizes human TF-1 cells, which proliferate in response to IL13. In this assay, TF-1 cells are cultured with serial dilutions of the recombinant IL13, and proliferation is measured after 48-72 hours using methods such as MTT/XTT or [3H]-thymidine incorporation. An ED50 of less than 5 ng/ml indicates fully active protein, corresponding to a specific activity of >2.0×10^5 IU/mg .

• Eotaxin induction: Measuring eotaxin levels in serum or BAL fluid provides an in vivo readout of IL13 activity. Anti-IL13 treatment in macaque models has been shown to reduce eotaxin concentrations following allergen challenge, indicating that eotaxin can serve as a pharmacodynamic marker of IL13 activity .

• Receptor binding assays: While not explicitly mentioned in the search results, receptor binding assays utilizing purified IL13 receptors (IL-13Rα1 and IL-13Rα2) can provide direct measurement of binding affinities and help distinguish specific from non-specific effects in complex biological systems.

• Downstream signaling markers: Assessment of STAT6 phosphorylation and other IL13-specific signaling events in target cells provides a mechanistic readout of IL13 activity that complements functional assays.

How does IL13 interact with immune cell subsets in Rhesus macaque disease models?

IL13 demonstrates complex interactions with various immune cell subsets in Rhesus macaque disease models, with implications for understanding both normal immunity and pathological conditions:

What are the challenges in translating IL13-focused research from Rhesus macaque models to human applications?

Translating IL13-focused research from Rhesus macaque models to human applications faces several significant challenges: