Recombinant Human IL23A & IL12B Heterodimer Protein (Active)

How is Recombinant Human IL23A & IL12B Heterodimer Protein produced in laboratory settings?

The production of Recombinant Human IL23A & IL12B Heterodimer Protein typically involves co-expression systems in mammalian cells, most commonly HEK293 cells . The process begins with constructing two separate expression plasmids: one encoding the IL23A (p19) sequence and another encoding the IL12B (p40) sequence . One or both subunits are typically fused with a polyhistidine tag to facilitate purification. As described in search result , "A DNA sequence encoding the IL23A (Q9NPF7) (Met 1-Pro 189) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the p40 subunit of human IL12, termed as IL12B (NP_002178.2) (Met 1-Ser 328) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 2."

These plasmids are co-transfected into mammalian host cells, allowing simultaneous expression of both subunits, which then assemble into the heterodimeric complex. Following expression, the protein is purified using affinity chromatography (typically utilizing the His-tag), followed by additional purification steps to achieve high purity (>90%) . The purified protein is then typically lyophilized in a buffer containing stabilizers like trehalose to maintain structural integrity during storage .

What functional assays are used to verify the activity of Recombinant IL23A & IL12B Heterodimer?

Multiple complementary assays are employed to verify the biological activity of Recombinant IL23A & IL12B Heterodimer:

• Receptor binding assays: These measure the heterodimer's ability to bind its receptors, including biotinylated recombinant human IL12RB1 in a functional ELISA . Typically, the heterodimer is immobilized at 10 μg/ml and its binding to human IL23R-Fc is quantified, with expected EC50 values of 0.28-0.66 μg/ml .

• Cynomolgus receptor binding: Similar assays measuring binding to Cynomolgus IL23R-Fc, with EC50 values of 0.14-0.35 μg/ml .

• Cellular functional assays: These assess the heterodimer's ability to induce IL17 secretion by mouse splenocytes, with typical ED50 values ranging from 0.5-20 ng/mL . As noted in search result : "Measured by its ability to induce IL17 secretion by mouse splenocytes. The ED50 for this effect is 4-20 ng/mL."

• Structural verification: SDS-PAGE analysis under reducing conditions confirms the presence of both subunits at their expected molecular weights, typically showing bands at 22-23 kDa (IL23A) and 40-45 kDa (IL12B) .

These assays collectively confirm both the structural integrity and functional activity of the recombinant heterodimer.

How do glycosylation patterns affect the biological activity of IL23A & IL12B Heterodimer?

Glycosylation patterns significantly impact the biological activity of IL23A & IL12B Heterodimer through multiple mechanisms. Both subunits undergo N-linked glycosylation, which contributes to their observed molecular weights being higher than calculated values . This is evident from SDS-PAGE analyses showing that IL23A migrates at 22-23 kDa (versus calculated 19-20 kDa) and IL12B at 40-45 kDa (versus calculated 34-36 kDa) .

Glycosylation influences:

• Protein folding and heterodimer formation: Proper glycosylation is essential for maintaining the three-dimensional conformation of both subunits, directly impacting their ability to form stable heterodimers.

• Receptor binding kinetics: Glycan structures modulate the interaction with IL23R and IL12RB1, affecting binding affinity and downstream signaling potency.

• Protein stability and half-life: Glycosylation affects proteolytic resistance and circulation time, influencing the duration of biological activity.

The expression system used for recombinant protein production is crucial, as different cell types produce proteins with distinct glycosylation patterns. This explains why HEK293 cells are commonly used for producing this heterodimer, as they provide human-like glycosylation patterns .

What are the important differences between human and cynomolgus IL23A & IL12B heterodimers in research applications?

Understanding the differences between human and cynomolgus IL23A & IL12B heterodimers is critical for translational research, particularly when developing therapeutic agents:

These differences highlight the importance of selecting appropriate models for studying IL-23 biology and evaluating IL-23-targeting therapeutics.

How stable is Recombinant Human IL23A & IL12B Heterodimer Protein under various storage conditions?

The stability of Recombinant Human IL23A & IL12B Heterodimer Protein varies significantly under different storage conditions:

• Lyophilized state: In lyophilized form with appropriate cryoprotectants (typically trehalose or mannitol with Tween 80), the heterodimer remains stable for up to 12 months when stored at -20°C to -80°C . As stated in search result : "Generally, lyophilized proteins are stable for up to 12 months when stored at -20 to -80°C."

• Reconstituted solution: Once reconstituted, stability decreases dramatically. At 4-8°C, the reconstituted protein typically maintains activity for only 2-7 days . For longer storage, aliquoting and storing at temperatures below -20°C is recommended, where stability extends to approximately 3 months.

• Formulation: The lyophilized protein is typically formulated with stabilizers: "Lyophilized from sterile PBS, pH 7.4. Normally 5% - 8% trehalose, mannitol and 0.01% Tween 80 are added as protectants before lyophilization" .

• Shipping conditions: The product is typically shipped as a lyophilized powder with ice packs to maintain low temperature during transport .

This stability profile highlights the importance of proper handling and storage to maintain the structural integrity and biological activity of the heterodimer.

What reconstitution protocols are recommended for maintaining IL23A & IL12B heterodimer functional integrity?

Proper reconstitution is critical for maintaining IL23A & IL12B heterodimer functional integrity:

• Temperature equilibration: The lyophilized protein should be equilibrated to room temperature before opening to prevent condensation.

• Reconstitution buffer: Use sterile PBS, pH 7.4, as indicated in search results : "Lyophilized from 0.22 μm filtered solution in PBS, pH7.4 with trehalose as protectant."

• Sterile technique: Use sterile techniques and preferably perform reconstitution in a biological safety cabinet to prevent contamination.

• Gentle mixing: Avoid vigorous vortexing, which can disrupt the heterodimeric structure. Instead, use gentle inversion or rotation until the solution appears homogeneous.

• Aliquoting: Immediately divide the reconstituted protein into single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise structural integrity.

• Storage of reconstituted protein: As noted in search result : "Reconstituted protein solution can be stored at 4-8°C for 2-7 days. Aliquots of reconstituted samples are stable at < -20°C for 3 months."

• Validation: After reconstitution, it is advisable to verify activity using one of the functional assays described previously to confirm that the reconstitution process has preserved biological activity.

These protocols should be optimized for specific research applications, particularly for experiments requiring high reproducibility.

How do polymorphisms in IL12B and IL23A genes affect protein structure and function in inflammatory disease models?

Polymorphisms in IL12B and IL23A genes can significantly alter protein structure and function in inflammatory diseases, as evidenced by studies in systemic lupus erythematosus (SLE) and other conditions:

• Promoter region variations: The study in search result investigated "IL12Bpro and IL12B 3′ untranslated region (UTR)" polymorphisms in SLE patients, examining their potential impact on disease susceptibility. These promoter variants can affect transcription and expression levels of the IL12B subunit.

• Functional impact: While the specific study in search result concluded that "polymorphisms located in IL12B, IL12RB1 and IL23A genes may not play a relevant role in the susceptibility or severity of SLE in the Spanish population," other research has found associations between these polymorphisms and inflammatory conditions.

• Receptor interaction effects: As noted in search result , "signaling through IL-23R may potentially promote IL-12 production, whereas signaling through IL-12Rβ2 suppresses IL-1β and IL-23." Polymorphisms affecting receptor structures could therefore alter this regulatory balance.

• Dichotomous expression patterns: Search result mentions "a dichotomous pattern of expression for IL-12 and IL-23 receptors in both mouse and humans," suggesting that immune cells involved in different responses may be regulated by distinct genetic factors.

Understanding these genetic variations is essential when designing and interpreting experimental models of inflammatory diseases, as they may influence cytokine production, receptor binding, and downstream signaling pathways.

What challenges exist in studying IL23A & IL12B heterodimer interactions with receptors in different tissue microenvironments?

Studying IL23A & IL12B heterodimer interactions with receptors across various tissue microenvironments presents several significant challenges:

• Subunit competition and alternative complexes: As described in search result , IL12B can form homodimers (IL-12p80) or combine with IL12A to form IL-12, while "IL-23p19 can interact with Ebi3." These alternative interactions compete with IL23A & IL12B heterodimer formation and complicate the interpretation of experimental results.

• Complex regulation: The study cited in search result notes that "IL-12 and IL-23 can also be regulated both genetically and epigenetically," with factors like phosphatase 2A negatively regulating IL-23 but not IL-12 in LPS-stimulated dendritic cells.

• Opposing functional effects: Search result highlights that "IL-4 had opposing effects on the production of either IL-12 or IL-23 in which it promoted the IL-12-producing capacity of DCs while abrogating IL-23 production." Such opposing regulation makes it difficult to predict net effects in complex tissue environments.

• Shared subunits and receptor components: The IL12B subunit is shared between IL-12 and IL-23, and IL12RB1 is a common receptor component. As noted in search result , "Free IL-12p40 exists in either homodimeric IL-12p80 or monomeric IL-12p40 forms in mice and human, and can act as natural antagonists of IL-12 and IL-23 by competing for binding to IL-12Rβ1."

These challenges necessitate sophisticated experimental approaches to distinguish heterodimer-specific effects from those of individual subunits or alternative complexes in physiologically relevant tissue contexts.

How can contradictory roles of IL23A & IL12B heterodimer in disease models be methodologically investigated?

The seemingly contradictory roles of IL23A & IL12B heterodimer in disease models require sophisticated methodological approaches:

• Addressing nonphysiological expression: Search result notes that "Although these data appear contradictory at first, the caveat of most of these experiments is that the expression of IL-23 is in a nonphysiological manner." This highlights the importance of using models with physiologically relevant expression patterns and levels.

• Studying regulatory interactions: As described in search result , IL-23 may antagonize IL-12-induced IFN-γ secretion, while signaling through IL-23R may potentially promote IL-12 production. These complex interactions require careful experimental design with appropriate controls for each pathway.

• Distinguishing genetic vs. epigenetic regulation: Search result mentions that "IL-12 and IL-23 can also be regulated both genetically and epigenetically," necessitating approaches that can separate these regulatory mechanisms.

• Investigating dichotomous receptor patterns: The "dichotomous pattern of expression for IL-12 and IL-23 receptors" mentioned in search result suggests that cell-specific targeting approaches would be valuable for clarifying the contradictory roles.

• Accounting for alternative complexes: Search result notes that "IL-23p19 can interact with Ebi3," suggesting the need for experimental designs that can distinguish between effects of canonical heterodimers versus alternative complexes.

These methodological considerations help explain apparently contradictory findings and emphasize the importance of context-dependent interpretation of IL23A & IL12B heterodimer functions in disease models.

How do post-translational modifications influence the functional activity of IL23A & IL12B Heterodimer?

Post-translational modifications significantly impact the functional activity of IL23A & IL12B Heterodimer:

• Glycosylation effects: Both subunits undergo extensive glycosylation, evidenced by their migration patterns in SDS-PAGE showing higher apparent molecular weights than calculated values . This glycosylation influences protein folding, stability, receptor binding, and biological activity.

• Disulfide bonding: The formation of proper disulfide bonds is critical for maintaining the heterodimeric structure and ensuring optimal receptor binding. Disruption of these bonds can lead to dissociation of the subunits and loss of biological activity.

• Processing by tissue-specific factors: Local tissue factors may modify the heterodimer in vivo, potentially generating variants with altered activity profiles. This may partly explain the context-dependent effects observed in different disease models .

• Potential regulatory modifications: While not explicitly detailed in the search results, research on related cytokines suggests that phosphorylation, ubiquitination, or other regulatory modifications may fine-tune heterodimer activity in response to the cellular environment.

Understanding these modifications is crucial when designing experiments to study IL23A & IL12B heterodimer function, as recombinant proteins from different sources or preparation methods may exhibit different modification patterns and consequently different functional properties.

How can researchers distinguish between effects of the IL23A & IL12B heterodimer versus those of individual subunits?

Distinguishing between effects of the intact IL23A & IL12B heterodimer versus individual subunits poses a significant experimental challenge:

• Shared subunit considerations: As noted in search result , "free IL-12p40 exists in either homodimeric IL-12p80 or monomeric IL-12p40 forms in mice and human, and can act as natural antagonists of IL-12 and IL-23 by competing for binding to IL-12Rβ1." This highlights the complexity of interpreting experiments where IL12B may exist in multiple forms.

• Alternative complex formation: Search result mentions that "IL-23p19 can interact with Ebi3," indicating that IL23A can potentially form alternative complexes beyond the canonical heterodimer with IL12B.

• Receptor-based discrimination: The study cited in search result describes "a dichotomous pattern of expression for IL-12 and IL-23 receptors," suggesting that receptor-specific approaches could help distinguish the effects of different cytokine complexes.

• Regulatory interactions: Search result notes that "signaling through IL-23R may potentially promote IL-12 production, whereas signaling through IL-12Rβ2 suppresses IL-1β and IL-23," highlighting the interconnected nature of these signaling pathways.

These complexities necessitate carefully designed experiments using specific neutralizing antibodies, receptor-deficient models, and structure-function mutants to delineate heterodimer-specific effects from those attributable to individual subunits or alternative complexes.