IL1RL1 Human, Sf9

What is IL1RL1 Human, Sf9 and what are its key characteristics?

IL1RL1 (Interleukin-1 receptor-like 1), also known as ST2, is a protein encoded by the IL1RL1 gene in humans. When expressed in Sf9 Baculovirus cells, it produces a single, glycosylated polypeptide chain containing 318 amino acids (positions 19-328) with an 8 amino acid His-tag at the C-terminus . The protein has a molecular mass of 36.0 kDa, though it appears as multiple bands between 40-57 kDa on SDS-PAGE due to glycosylation . IL1RL1 is a member of the Toll-like receptor superfamily based on its intracellular TIR domain, while its extracellular region consists of immunoglobulin domains . The recombinant protein is typically purified using proprietary chromatographic techniques and formulated in phosphate-buffered saline (pH 7.4) with 10% glycerol .

What are the known isoforms of IL1RL1 and their functional differences?

IL1RL1 exists in two primary isoforms with distinct biological functions: a soluble form (referred to as soluble ST2 or sST2) and a membrane-bound receptor form (referred to as the ST2 receptor or ST2L) . The membrane-bound form functions as a receptor for Interleukin-33 (IL-33), while the soluble form acts as a decoy receptor that can bind IL-33 and prevent it from interacting with ST2L . These isoforms play critical roles in cardiac function regulation, where the IL-33/ST2L signaling provides cardioprotective effects in response to cardiac stress or injury. The soluble ST2 counterbalances this protection by sequestering IL-33, thereby preventing its binding to ST2L . This interplay is particularly relevant in cardiovascular research, where elevated sST2 levels are associated with increased cardiac stress .

How does recombinant IL1RL1 Human, Sf9 differ from native human IL1RL1?

Recombinant IL1RL1 produced in Sf9 Baculovirus cells represents the extracellular domain (amino acids 19-328) of the full-length protein (556 amino acids) . This recombinant form includes an 8-amino acid His-tag at the C-terminus for purification purposes, which is not present in the native protein . While the native IL1RL1 has a calculated molecular weight of 63,358 Da, the recombinant form has a calculated mass of 36.0 kDa but appears larger (40-57 kDa) on SDS-PAGE due to post-translational modifications, particularly glycosylation . The insect cell expression system may produce glycosylation patterns that differ from mammalian-expressed proteins, potentially affecting certain functional properties while maintaining core biological activity. Despite these differences, the recombinant protein preserves the essential IL-33 binding capacity, as demonstrated by its ability to inhibit proliferation in D10.G4.1 mouse helper T cells with an ED50 of ≤10ng/ml in the presence of IL-33 .

What are the optimal experimental conditions for studying IL1RL1/IL-33 signaling pathways in vitro?

For studying IL1RL1/IL-33 signaling pathways in vitro, researchers should consider several methodological approaches. When designing experiments with recombinant IL1RL1, the protein solution (typically at 0.5 mg/ml) should be maintained in phosphate-buffered saline (pH 7.4) with 10% glycerol . For long-term stability, addition of a carrier protein (0.1% HSA or BSA) is recommended, and multiple freeze-thaw cycles should be avoided .

For functional assays, the biological activity of IL1RL1 can be measured by its ability to inhibit proliferation using D10.G4.1 mouse helper T cells in the presence of IL-33, with an expected ED50 of ≤10ng/ml . When studying receptor-ligand interactions, solid-phase binding assays can be employed, as demonstrated by immobilized Human IL-33 Protein binding to Biotinylated Human IL-1RL1 with a linear detection range of 0.3-10 ng/mL .

For MAP kinase activation studies, it's important to note that unlike other family members, IL1RL1 does not induce inflammatory responses through NF-κB activation, though it does activate MAP kinases . This distinct signaling characteristic should be considered when designing pathway analysis experiments and selecting appropriate readouts or markers.

How can researchers effectively differentiate between the biological effects of membrane-bound ST2L and soluble ST2 in experimental models?

Differentiating between the biological effects of membrane-bound ST2L and soluble ST2 requires careful experimental design. Researchers can employ several strategies:

• Selective expression systems: Using recombinant expression of either the membrane-bound or soluble form exclusively allows for isolated study of each isoform's effects.

• Neutralizing antibodies: Employing antibodies that specifically target either ST2L or sST2 can help discriminate their individual contributions.

• Domain-specific binding partners: Creating constructs that selectively interact with either isoform provides another approach to distinguish their effects.

• Cardiac stress models: In cardiovascular research, monitoring myocardial stretch-induced upregulation of the ST2 gene can provide insights into the physiological balance between soluble ST2 and the IL-33/ST2L signaling axis .

• Functional readouts: Assessing cardioprotective effects (associated with ST2L) versus inhibition of these effects (associated with sST2) through functional assays such as cell survival, hypertrophy, or fibrosis measurements.

When planning such experiments, it's crucial to recognize that binding of IL-33 to the ST2 receptor elicits cardioprotective effects, while soluble ST2 counteracts this by sequestering IL-33 and preventing its interaction with ST2L . This counterbalancing mechanism results in increased cardiac stress under conditions of elevated soluble ST2 levels.

What are the current challenges in interpreting IL1RL1 data across different experimental models and how can they be addressed?

Interpreting IL1RL1 data across experimental models presents several challenges that researchers should address methodically:

• Glycosylation heterogeneity: The recombinant IL1RL1 shows multiple bands (40-57 kDa) on SDS-PAGE due to variable glycosylation , which may affect protein-protein interactions. Researchers should employ deglycosylation assays or mass spectrometry to characterize glycosylation patterns when comparing across models.

• Expression system differences: IL1RL1 produced in Sf9 Baculovirus cells may have different post-translational modifications compared to mammalian expression systems. When comparing results, researchers should account for these system-specific variations by including appropriate controls.

• Isoform-specific effects: The distinct roles of soluble ST2 versus membrane-bound ST2L require careful experimental design to distinguish their individual contributions. Using isoform-specific detection methods or selective inhibition approaches can help resolve these differences.

• Context-dependent signaling: IL1RL1 interacts with multiple downstream molecules (MyD88, IRAK1, IRAK4, and TRAF6) , but these interactions may vary across cell types or experimental conditions. Pathway validation using multiple approaches is recommended.

• Differential expression in disease states: Elevated soluble ST2 levels in cardiac stress conditions may complicate interpretation of experimental interventions. Researchers should establish baseline levels and monitor dynamic changes rather than relying on single measurements.

To address these challenges, researchers should implement comprehensive controls, validate findings across multiple experimental systems, and employ complementary analytical techniques to build robust interpretations of IL1RL1-related data.

What are the optimal storage and handling conditions for IL1RL1 Human, Sf9 to maintain protein stability and activity?

For optimal stability and activity of IL1RL1 Human, Sf9, researchers should follow these evidence-based handling protocols:

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

• Long-term storage: Store frozen at -20°C for extended periods. For maximum stability, store in lyophilized state at -20°C or lower .

• Formulation stability: The protein solution (0.5 mg/ml) typically contains phosphate-buffered saline (pH 7.4) with 10% glycerol . For enhanced stability during long-term storage, addition of a carrier protein (0.1% HSA or BSA) is strongly recommended .

• Reconstitution: For lyophilized products, follow the specific reconstitution protocol provided in the Certificate of Analysis . Generally, reconstitute in sterile water or appropriate buffer.

• Freeze-thaw cycles: Multiple freeze-thaw cycles should be strictly avoided as they can significantly degrade protein quality and activity .

• Working solutions: When preparing dilutions for experiments, use fresh, sterile buffers and keep solutions on ice when possible.

• Quality control: Before experimental use, verify protein integrity through SDS-PAGE analysis, ensuring the characteristic multiple band pattern between 40-57 kDa under reducing conditions .

Following these handling guidelines will help maintain the structural integrity and functional activity of IL1RL1 Human, Sf9, ensuring reliable and reproducible experimental results.

What are the recommended methodologies for assessing IL1RL1 binding affinity to IL-33 and other potential interaction partners?

For rigorous assessment of IL1RL1 binding interactions, researchers should consider the following methodological approaches:

• Solid-phase binding assays: Immobilize Human IL-33 Protein (e.g., His-tagged) at 5 μg/mL and measure binding to Biotinylated Human IL-1RL1. This approach has demonstrated a linear detection range of 0.3-10 ng/mL for binding interactions .

• Surface Plasmon Resonance (SPR): For real-time, label-free analysis of binding kinetics between IL1RL1 and IL-33 or other partners. This allows determination of association and dissociation rate constants (kon and koff) and equilibrium dissociation constant (KD).

• Functional proliferation inhibition assays: Measure IL1RL1 activity by its ability to inhibit proliferation in D10.G4.1 mouse helper T cells in the presence of IL-33. The effective dose for 50% inhibition (ED50) should be ≤10ng/ml, providing a functional readout of binding .

• Co-immunoprecipitation: To investigate interactions between IL1RL1 and proposed signaling partners (MyD88, IRAK1, IRAK4, and TRAF6) in cell-based systems.

• SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering): For accurate molecular weight determination and complex formation analysis. This technique has been validated for IL1RL1 quality assessment with >90% purity .

• Competitive binding assays: To evaluate binding specificity and identify potential competitive inhibitors of the IL1RL1/IL-33 interaction.

When interpreting binding data, researchers should account for the glycosylation heterogeneity of IL1RL1 (appearing as multiple bands between 40-57 kDa on SDS-PAGE) , as this may influence binding characteristics in different experimental systems.

What analytical techniques are most effective for characterizing the structural and functional properties of recombinant IL1RL1 Human, Sf9?

A comprehensive characterization of recombinant IL1RL1 Human, Sf9 requires multiple complementary analytical techniques:

For structural studies, it's important to note that IL1RL1 contains immunoglobulin domains in its extracellular region , which should be preserved in the recombinant form. Functional characterization should consider that while IL1RL1 does not activate NF-κB (unlike other family members), it does activate MAP kinases , providing useful functional readouts for activity assessment.

What are the most common issues encountered when working with IL1RL1 Human, Sf9 in experimental settings and how can they be resolved?

Researchers frequently encounter several challenges when working with IL1RL1 Human, Sf9. Here are evidence-based approaches to troubleshoot these issues:

• Inconsistent protein migration on SDS-PAGE

  • Observation: Variability in banding pattern beyond the expected 40-57 kDa range .

  • Solution: This heterogeneity is primarily due to glycosylation. Standardize sample preparation conditions and consider deglycosylation treatments for more uniform analysis when required.

• Loss of activity during storage

  • Observation: Decreased biological function in inhibition assays with D10.G4.1 cells.

  • Solution: Store at +4°C only if using within 2-4 weeks; otherwise maintain at -20°C with 10% glycerol and a carrier protein (0.1% HSA or BSA) . Strictly avoid repeated freeze-thaw cycles .

• Insufficient binding in interaction studies

  • Observation: Weak or inconsistent binding to IL-33.

  • Solution: Verify protein integrity by SDS-PAGE before experiments. For binding assays, use freshly prepared protein and optimize buffer conditions. When immobilizing IL-33, a concentration of 5 μg/mL has been validated for linear binding detection in the range of 0.3-10 ng/mL .

• Variable functional activity

  • Observation: Inconsistent ED50 values in cell-based assays.

  • Solution: Standardize experimental conditions, particularly temperature and incubation times. Ensure D10.G4.1 cell viability and passage number consistency. The expected ED50 should be ≤10ng/ml with IL-33 .

• Protein aggregation

  • Observation: Visible particulates or precipitates in solution.

  • Solution: Centrifuge solutions briefly before use. Consider adding carrier proteins for stability, and avoid exposing to non-physiological conditions. Filter through 0.22 μm filters if necessary .

By implementing these targeted troubleshooting approaches, researchers can significantly improve the reliability and reproducibility of experiments involving IL1RL1 Human, Sf9.

How can researchers effectively design experiments to distinguish between IL1RL1-specific effects and non-specific experimental artifacts?

Designing experiments that clearly differentiate IL1RL1-specific effects from experimental artifacts requires methodical controls and validation approaches:

• Multiple negative controls:

  • Include structurally similar but functionally distinct proteins (e.g., other IL-1 receptor family members) to control for non-specific interactions.

  • Use denatured IL1RL1 to distinguish structure-dependent functions from non-specific effects.

  • Implement mock-transfected or untreated control groups in all cell-based experiments.

• Dose-response relationships:

  • Establish complete dose-response curves for IL1RL1 effects, rather than single-dose experiments.

  • Verify that effects follow expected pharmacological principles (e.g., saturation at higher concentrations).

  • The validated functional dose range for IL1RL1 with D10.G4.1 cells and IL-33 shows an ED50 of ≤10ng/ml .

• Orthogonal validation:

  • Confirm findings using multiple, methodologically distinct approaches.

  • Complement binding studies with functional assays and vice versa.

  • Verify protein-protein interactions through different techniques (e.g., SPR, co-IP, ELISA).

• Genetic approaches:

  • Use genetic knockdown/knockout of IL1RL1 to confirm specificity of observed effects.

  • Employ domain-specific mutations to map functional regions mediating specific interactions.

  • Consider the known interactions with MyD88, IRAK1, IRAK4, and TRAF6 as positive controls for specific binding.

• Isoform controls:

  • Compare effects of membrane-bound ST2L versus soluble ST2 to distinguish their specific contributions .

  • Use isoform-selective reagents to differentiate their individual effects.

• Biological relevance validation:

  • Correlate in vitro findings with known physiological roles, such as the cardioprotective effects of IL-33/ST2L signaling versus the counterbalancing effects of soluble ST2 .

  • Consider the established role in T helper cells type 2 (Th2 cells) function as a reference point for immunological experiments.

Implementing these control strategies will significantly enhance the specificity and reliability of IL1RL1-related research findings.

What are the current gaps in our understanding of IL1RL1 function and what experimental approaches might address these gaps?

Despite significant advances in IL1RL1 research, several critical knowledge gaps remain that require innovative experimental approaches:

• Isoform-specific signaling networks

  • Knowledge gap: The differential signaling pathways activated by membrane-bound ST2L versus effects of soluble ST2 remain incompletely characterized.

  • Experimental approach: Phosphoproteomics and interactome analysis comparing cells expressing exclusively ST2L versus those exposed to soluble ST2, coupled with temporal signaling cascade mapping.

• Tissue-specific functions

  • Knowledge gap: While cardiac roles are well-studied , the function of IL1RL1 in other tissues remains less defined.

  • Experimental approach: Tissue-specific conditional knockout models coupled with comprehensive phenotyping and single-cell transcriptomics to identify cell type-specific responses.

• Regulatory mechanisms controlling isoform expression

  • Knowledge gap: The molecular switches determining the balance between soluble and membrane-bound forms are poorly understood.

  • Experimental approach: CRISPR-based promoter/enhancer screening to identify regulatory elements controlling differential expression, combined with RNA-binding protein identification.

• Structural determinants of IL-33 binding specificity

  • Knowledge gap: The precise structural features of IL1RL1 that confer selectivity for IL-33 versus other cytokines remain unclear.

  • Experimental approach: Cryo-EM or X-ray crystallography of the IL1RL1/IL-33 complex, complemented by directed mutagenesis of the binding interface.

• Non-IL-33 ligands and binding partners

  • Knowledge gap: Whether IL1RL1 interacts with molecules beyond IL-33 and known signaling adaptors (MyD88, IRAK1, IRAK4, TRAF6) remains uncertain.

  • Experimental approach: Unbiased interactome analysis using BioID or APEX proximity labeling, followed by validation of novel interactions.

• Therapeutic targeting strategies

  • Knowledge gap: Optimal approaches to modulate the IL-33/IL1RL1 axis for therapeutic benefit remain to be determined.

  • Experimental approach: Development of isoform-selective modulators (e.g., antibodies or small molecules) that can specifically enhance or inhibit either ST2L or soluble ST2 function.

Addressing these knowledge gaps will require integration of structural biology, systems biology, and in vivo models to comprehensively map IL1RL1 function across physiological and pathological contexts.