Recombinant Human Interleukin-13 protein (IL13), partial (Active)

What is the molecular structure of human IL-13?

Human IL-13 belongs to the short-four-helix-bundle cytokine family. The solution structure determined by multidimensional NMR reveals a left-handed four-helix bundle topology with significant structural similarity to IL-4, which explains their overlapping biological functions . The protein contains two disulfide bonds (Cys29–Cys57 and Cys45–Cys71) that are essential for maintaining its tertiary structure and biological activity . The mature protein consists of amino acids 25-146, with a calculated molecular weight of approximately 12.3 kDa .

What are the primary biological functions of IL-13?

IL-13 is a pleiotropic Th2 cytokine that functions in multiple biological processes:

• Plays central roles in allergic inflammation and immune responses to parasitic infections

• Synergizes with IL-2 in regulating interferon-gamma synthesis

• Stimulates B-cell proliferation and activation of eosinophils, basophils, and mast cells

• Controls IL-33 activity by modulating the production of transmembrane and soluble forms of IL1RL1

• Antagonizes Th1-driven proinflammatory immune responses by downregulating synthesis of IL1, IL6, IL10, IL12, and TNF-alpha through partial suppression of NF-kappa-B

• Induces expression of vascular cell adhesion protein 1 (VCAM1) in endothelial cells, facilitating eosinophil recruitment

• Acts as a central mediator in allergic asthma pathophysiology

How does IL-13 signaling work at the cellular level?

IL-13 exerts its biological effects through a heterodimeric receptor complex. The primary signaling pathway involves:

• Binding to the IL-13Rα1 chain

• Recruitment of the IL-4R chain to form a heterodimeric complex

• Activation of JAK1 and TYK2 tyrosine kinases

• Phosphorylation and activation of STAT6 transcription factor

Additionally, IL-13 can bind to IL-13Rα2, which primarily functions as a high-affinity decoy receptor that mediates internalization and depletion of extracellular IL-13 . Recent evidence suggests that IL-13Rα2 may also contribute to signaling cascades under specific conditions .

What expression systems are commonly used for recombinant human IL-13 production?

Recombinant human IL-13 can be produced in several expression systems:

• Mammalian cell expression (HEK293 cells):

  • Advantages: Proper folding, post-translational modifications

  • Produces protein with ≥95% purity and ≤0.005 EU/μg endotoxin levels

  • Typically yields full-length protein (amino acids 25-146)

• E. coli expression:

  • Advantages: Higher yield, cost-effective

  • Typically produces partial protein (amino acids 35-146)

  • Requires refolding protocols to achieve proper tertiary structure

• CHO cell expression:

  • Provides consistent glycosylation patterns

  • Yields highly active protein (activity ED₅₀< 15 ng/ml in TF-1 cell proliferation assays)

What are the challenges in purification and refolding of recombinant IL-13?

When expressed in E. coli, IL-13 often localizes to inclusion bodies, necessitating solubilization and refolding. A validated protocol includes:

• Expression as a fusion protein (e.g., with maltose-binding protein)

• Purification of inclusion bodies using denaturing conditions

• Refolding through controlled dilution in buffer containing appropriate redox agents

• Confirmation of correct disulfide bond formation (Cys29–Cys57 and Cys45–Cys71)

This process typically yields approximately 2 mg of bioactive protein per liter of bacteria grown in minimal media . Critical factors affecting refolding efficiency include:

• Protein concentration during refolding

• Redox buffer composition and pH

• Temperature and duration of refolding

• Presence of stabilizing additives

How is the activity of recombinant IL-13 measured and what benchmarks indicate proper folding?

Activity of recombinant human IL-13 is commonly measured using:

• TF-1 cell proliferation assay:

  • High-quality preparations show ED₅₀ values between 1.5-15 ng/ml

  • This corresponds to a specific activity of >6.7×10⁵ units/mg

• STAT6 phosphorylation assay:

  • Active IL-13 stimulates STAT6 phosphorylation in cells expressing IL-13Rα1/IL-4R

  • EC₅₀ values of 0.75 ng/ml for human IL-13 and 10 ng/ml for macaque IL-13

• Eotaxin production assay in normal human lung fibroblasts:

  • EC₅₀ values of 7.5 ng/ml for human IL-13

How should recombinant IL-13 be stored and handled to maintain optimal activity?

For optimal stability and activity retention:

• Storage conditions:

  • Store lyophilized protein at -20°C to -80°C

  • Reconstituted protein should be stored at -80°C in single-use aliquots

  • Avoid repeated freeze-thaw cycles

• Reconstitution protocol:

  • Reconstitute in sterile buffer (PBS or similar)

  • For low concentration solutions (<0.1 mg/ml), add carrier protein (0.1-0.5% BSA) to prevent adsorption

  • Gentle mixing, avoid vortexing which can cause protein denaturation

• Working solution preparation:

  • Dilute stock solutions immediately before use

  • Use polypropylene tubes to minimize protein adsorption

  • Prepare solutions on ice when possible

What are the optimal conditions for using IL-13 in cell culture experiments?

When designing experiments with IL-13 stimulation:

• Effective concentration range:

  • For most immune cell studies: 5-50 ng/ml

  • For EC cell studies: 10 ng/ml showed significant effects

  • For neuronal studies: 10 ng/ml induced significant signaling changes

• Treatment duration:

  • Acute responses: 30 minutes to 4 hours

  • Gene expression changes: 4-24 hours

  • Phenotype alterations: 24-72 hours

• Cell-specific considerations:

  • TF-1 cells show proliferative responses within 24-48 hours

  • BON cells (enterochromaffin model) show hyperplasia after 24 hours

  • Neuronal cells exhibit phosphorylation changes within 1-3 hours

How can IL-13 be used in studying immune cell responses?

IL-13 is valuable for modeling Th2-type immune responses:

• Macrophage alternative activation:

  • Typically use 10-20 ng/ml for 24-48 hours

  • Measure markers like arginase-1, CD206, and YM1/2

  • IL-13 may be used alone or in combination with IL-4

• B-cell studies:

  • IL-13 stimulates B-cell proliferation and IgE class switching

  • Typical protocols use 5-20 ng/ml for 3-5 days

  • Measure outcomes through flow cytometry and ELISA

• Mast cell and eosinophil activation:

  • IL-13 extends survival and enhances activation

  • In mouse models, IL-13 treatment (10 ng/ml) increased mast cell numbers four-fold in skin tissue

How can recombinant IL-13 be used in neurobiological research?

Recent research has revealed important neurobiological roles for IL-13:

• Synaptic localization:

  • IL-13 and IL-13Rα1 are localized to synapses in cortical neurons

  • Approximately 70% of VGLUT+ synapses show immunoreactivity for IL-13 or IL-13Rα1

  • IL-13 protein is enriched in synaptic vesicle fractions similar to synaptophysin

• Signaling effects in neurons:

  • IL-13 (10 ng/ml for 1-3 hours) triggers phosphorylation of glutamate receptors:

    • NMDAR1 (S897)

    • AMPAR1 (S849 and S863)

    • CaMKII (T286 and T305)

    • TrkB (Y515 and Y705)

  • Induces immediate-early gene expression (c-fos, fos-B, egr-1)

• Experimental protocols:

  • For neuronal cultures: use 10 ng/ml for 1-3 hours

  • Assess phosphorylation by western blot or immunofluorescence

  • Measure gene expression changes via qPCR

What are the considerations when using IL-13 in animal models?

When designing in vivo experiments:

• Dosing considerations:

  • For mouse models: 0.2-2 μg/mouse (intraperitoneal or intranasal)

  • Administration frequency typically ranges from daily to twice weekly

  • In IL-13-deficient mice, restoration with recombinant IL-13 (5 days of treatment) significantly increased enterochromaffin cell numbers and 5-HT content in the colon

• Species cross-reactivity:

  • Human and mouse IL-13 are cross-species reactive

  • Human IL-13 binds to macaque IL-13Rα1 and IL-13Rα2 with IC₅₀ values of 64 and 9.5 ng/ml, respectively

• Transgenic approaches:

  • Targeted expression in specific tissues (e.g., skin) can induce inflammatory phenotypes

  • In skin-specific IL-13 transgenic mice, the cytokine induced a chronic pruritic inflammatory phenotype resembling atopic dermatitis

What modifications improve IL-13 stability or activity for specialized applications?

Advanced modifications include:

• Site-directed mutagenesis:

  • The R144Q mutation is commonly used in partial recombinant proteins (aa 35-146)

  • Altering disulfide bonds can significantly impact activity:

    • The C46–C99 disulfide bond is essential for IL-13 activity

    • Selective reduction of this bond by thioredoxin 1 inactivates the cytokine

• Fusion proteins:

  • IL-13-PE (IL-13 fused to Pseudomonas exotoxin) targets IL-13 receptor-expressing tumor cells

  • Fc-fusion proteins increase half-life for in vivo applications

• Isotopic labeling:

  • ¹⁵N-labeled IL-13 can be produced for NMR structural studies

  • Expression systems using HEK293 or E. coli in minimal media yield approximately 2 mg/L

What are common issues when working with recombinant IL-13 and how can they be resolved?

How can researchers distinguish between different IL-13 signaling pathways in experimental systems?

To differentiate IL-13 signaling pathways:

• Receptor-specific approaches:

  • Use blocking antibodies against IL-13Rα1 vs. IL-13Rα2

  • Employ siRNA knockdown of specific receptor components

  • Use cells from receptor knockout models

• Pathway inhibitors:

  • JAK inhibitors (e.g., Tofacitinib) for canonical signaling

  • STAT6 inhibitors for transcriptional effects

  • MAPK/ERK inhibitors for non-canonical pathways

• Readout selection:

  • STAT6 phosphorylation (Y641) for canonical pathway

  • ERK1/2 phosphorylation for alternative signaling

  • In neurons, CREB phosphorylation occurs within 1-3 hours of IL-13 treatment

How can researchers assess IL-13 interactions with other cytokines in complex experimental systems?

For investigating cytokine networks:

• Co-stimulation experiments:

  • Compare IL-13 alone vs. IL-13+IL-4 (synergistic effects)

  • Test IL-13 in presence of Th1 cytokines (antagonistic effects)

  • Examine IL-13 and IL-33 cross-regulation

• Neutralization strategies:

  • Sequential neutralization of cytokines

  • Time-course analysis of cytokine production

  • Receptor blockade experiments

• Advanced techniques:

  • Cytokine secretion assays (e.g., ELISpot)

  • Multiplex cytokine profiling

  • Single-cell RNA sequencing for heterogeneous responses

What are emerging applications for recombinant IL-13 in neuroscience research?

Recent discoveries highlight promising areas:

• Synaptic plasticity and learning:

  • IL-13 may be involved in neuromodulation and synaptic plasticity underlying spatial memory and learning

  • The protein triggers large-scale phosphorylation of glutamate receptors and presynaptic proteins

• Neuroinflammation and traumatic brain injury:

  • Increased IL-13 is a hallmark of traumatic brain injury in mice and humans

  • Research could focus on therapeutic potential in neurological disorders

• Neural-immune interactions:

  • IL-13 may serve as a communication mediator between immune and nervous systems

  • Potential role in neuro-immune disorders warrants investigation

What novel methodologies are being developed for studying IL-13 signaling dynamics?

Emerging approaches include:

• Real-time biosensors:

  • FRET-based reporters for detecting IL-13/receptor interactions

  • Optogenetic tools for spatiotemporal control of IL-13 release

• High-throughput screening:

  • CRISPR-based screens to identify novel IL-13 signaling components

  • Small molecule libraries to discover pathway modulators

• Advanced imaging:

  • Super-resolution microscopy to visualize receptor clustering

  • Intravital imaging to track IL-13 signaling in vivo

How might understanding IL-13 redox susceptibility inform therapeutic development?

The discovery that IL-13 is susceptible to thioredoxin-mediated inactivation opens new avenues:

• Redox regulation mechanisms:

  • Thioredoxin 1 preferentially inactivates IL-4 over IL-13 through reduction of the C46–C99 disulfide bond

  • This provides a potential mechanism for differential regulation of these related cytokines

• Engineered variants:

  • Designing redox-resistant IL-13 variants for enhanced stability

  • Creating redox-sensitive variants for controlled release systems

• Therapeutic targeting:

  • Inhibitors of secreted thioredoxin (e.g., NP161) enhance alternatively activated macrophage cytokine outputs

  • This represents a novel pharmacologically promising immunomodulatory mechanism