IL 4 Human, Yeast

What are the essential components of a purification protocol for recombinant human IL-4 expressed in yeast?

An effective purification protocol for yeast-expressed human IL-4 typically involves a multi-step process. Based on established protocols, this includes:

• Initial separation: Passing crude cell-free conditioned medium over a concanavalin A-Sepharose affinity column, which binds glycoproteins .

• Ion exchange chromatography: Using S-Sepharose Fast Flow cation exchange to separate proteins based on charge .

• High-performance liquid chromatography: Final purification using C18 reverse-phase HPLC to obtain highly purified IL-4 .

This protocol yields highly purified IL-4 (0.3-0.4 mg per liter of culture) with a recovery of approximately 51% . The methodology can be adjusted based on specific expression constructs and fusion tags. When working with glycoengineered IL-4 variants, additional steps may be necessary to process or analyze the glycan structures.

How do researchers confirm the structural integrity of yeast-produced human IL-4?

Confirming the structural integrity of yeast-produced human IL-4 requires multiple analytical approaches:

• Mass spectrometry: Thermospray liquid chromatography-mass spectrometry can confirm both the C-terminal N-glycosylation status and the authenticity of the N-terminus .

• Disulfide bond analysis: Thiol titration to verify the absence of free cysteine residues, indicating proper formation of the three disulfide groups critical for IL-4 structure .

• Biological activity assays: Validation through multiple functional assays, including B-cell co-stimulator assays, T-cell proliferation assays, and induction of CD23 expression on tonsillar B-cells .

• Receptor binding studies: Radioiodinated IL-4 can be used in equilibrium binding studies to confirm proper receptor interactions, with properly folded IL-4 showing high-affinity binding (Kd = 100 pM) .

The presence of all three native disulfide bonds is particularly important for maintaining IL-4's correct tertiary structure and biological function.

How can researchers design IL-4 antagonists through site-specific modifications in yeast expression systems?

Designing IL-4 antagonists through site-specific modifications involves strategic alterations to create molecules that bind to IL-4Rα but prevent recruitment of secondary receptor chains. This can be achieved through several approaches:

• Mutation-based strategies: Introducing mutations that enhance IL-4Rα binding while disrupting receptor heterodimerization. For example, the F82D mutation enhances affinity for IL-4Rα approximately 3-fold, allowing IL-4 antagonists to more effectively compete with endogenous wild-type IL-4 .

• Glycoengineering approaches: Introducing glycan moieties at specific positions to create steric hindrance:

  • Chemical glycosylation: Direct coupling of thiol-carbohydrates to cysteine variants during refolding .

  • Site-specific glycosylation: Introducing glycans at positions that don't interfere with IL-4Rα binding but block recruitment of secondary receptor chains .

• Combined strategies: For example, the IL-4 F82D R121C variant can be chemically glycosylated to produce antagonists with inhibitory constants (Ki) of 14.6-17.4 pM in TF-1 cells, which is more potent than the benchmark antagonist Pitrakinra (Ki = 98.3 pM) .

These approaches require careful consideration of IL-4's structure-function relationships to maintain primary receptor binding while specifically disrupting secondary interactions required for signaling.

What methods enable selective chemical glycosylation of IL-4 produced in yeast systems?

Selective chemical glycosylation of IL-4 can be achieved through several methodological approaches:

• Site-directed mutagenesis: Introducing unique cysteine residues at specific positions (e.g., R121C) that can serve as attachment points for glycan moieties .

• Direct coupling during refolding: A streamlined approach involves:

  • Denaturing the IL-4 protein using GuHCl

  • Adding the denatured protein to refolding buffer containing thiol-glycans

  • Allowing conjugation to occur during the refolding process at 4°C for 72 hours

  • Dialyzing the refolding mixture against ammonium acetate

This direct coupling method offers nearly 100% conjugation efficiency without requiring enzymatic deglutathionylation or thiol-activation steps .

• Bifunctional crosslinker approach: Although more complex, this involves:

  • Using succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) as a crosslinker

  • Creating a glucosamine-SMCC conjugate

  • Reacting this conjugate with enzymatically deglutathionylated IL-4 F82D R121C

  • Purifying the reaction product via RP-HPLC

Each method has distinct advantages, with the direct coupling approach offering superior efficiency for most research applications.

How do researchers characterize the receptor binding properties of modified IL-4 variants expressed in yeast?

Characterizing receptor binding properties of modified IL-4 variants involves several complementary analytical approaches:

• Surface Plasmon Resonance (SPR): This technique allows determination of binding kinetics and affinity constants. For example, glycoengineered IL-4 variants have shown KD values of 16.6 ± 0.4 pM, indicating high-affinity binding despite modifications .

• Radioligand binding assays: 125I-labeled IL-4 can be used in equilibrium binding studies with receptor-expressing cells (e.g., Raji B-cells) to determine binding parameters. Properly folded recombinant IL-4 typically shows high-affinity binding (Kd = 100 pM) and can identify approximately 1100 receptors per cell .

• Receptor-ligand cross-linking studies: This approach can visualize and characterize the cell-surface receptor complex, which for IL-4 appears as a single receptor with an apparent molecular mass of 124 kDa .

• Functional reporter assays: Cell-based assays using TF-1 cell proliferation or HEK-Blue SEAP reporter systems can indirectly assess receptor interaction through downstream signaling events .

These methods collectively provide a comprehensive profile of how modifications affect receptor binding dynamics, informing rational design of IL-4 variants with desired properties.

What are the key considerations for optimizing disulfide bond formation in yeast-expressed human IL-4?

Optimizing disulfide bond formation in yeast-expressed human IL-4 requires careful attention to several factors:

• Redox environment: Using a glutathione-based redox couple (oxidized and reduced glutathione) during in vitro refolding facilitates proper disulfide formation. The correct ratio and concentration of these components are critical for maximizing correct folding .

• Protection of unpaired cysteines: In IL-4 variants with introduced cysteines (e.g., R121C), the unpaired cysteine often forms a mixed disulfide with glutathione during refolding. This "protection" group prevents non-specific reactions but must be removed before further modifications .

• Selective deglutathionylation: For variants with added cysteines, enzymatic approaches for removing glutathione adducts are preferred over chemical reduction, as chemical reducing agents can also cleave IL-4's native disulfide bonds, leading to denaturation and precipitation .

• Refolding conditions: Optimizing parameters such as protein concentration (typically 1-1.5 mg/mL), temperature (4°C for 72 hours), pH (around 8.0), and the inclusion of folding enhancers like arginine (1M) significantly improves folding yield and disulfide bond formation .

• Verification: Thiol titration to confirm the absence of free cysteine residues, indicating all cysteines have formed disulfide bonds or are otherwise protected .

Proper formation of IL-4's three disulfide groups is essential for maintaining its tertiary structure and biological activity.

How can researchers address the challenges of glycosylation heterogeneity in yeast-expressed IL-4?

Addressing glycosylation heterogeneity in yeast-expressed IL-4 requires strategic approaches:

• Engineered N-glycosylation sites: Introducing specific N-glycosylation motifs (Asn-X-Ser/Thr) at selected positions can direct glycosylation. Research has identified promising sites where glycosylation doesn't interfere with IL-4Rα binding, including positions Q20N, T28N, and K61N .

• Glycoengineering yeast strains: Using modified yeast strains with altered glycosylation pathways can produce more homogeneous glycan structures or glycoforms that more closely resemble mammalian glycosylation.

• Enzymatic homogenization: Treatment with specific glycosidases can trim heterogeneous glycans to create more uniform structures. PNGase F can be used to verify N-glycosylation by enzymatic hydrolysis of N-linked oligosaccharides .

• Chemical glycosylation: For precise control over glycan structure, chemical approaches using defined glycan moieties can be coupled to specific sites (e.g., introduced cysteines) during or after protein folding .

• Analytical characterization: Comprehensive analysis using mass spectrometry, SDS-PAGE, and lectin binding assays can identify and quantify glycoform distributions, guiding optimization efforts .

Researchers should note that yeast-derived IL-4 typically contains poly-mannose glycans without terminal sialic acid residues, unlike the complex or hybrid N-glycans with sialic acid termini produced in mammalian cells, which may affect properties like proteolytic stability .

What strategies can improve the yield and solubility of recombinant human IL-4 in yeast expression systems?

Improving yield and solubility of recombinant human IL-4 in yeast expression systems can be achieved through multiple strategies:

• Secretion signal optimization: Using the yeast prepro alpha-mating factor sequence as a fusion partner promotes efficient secretion of mature IL-4 into the culture medium, facilitating purification and typically improving solubility .

• Codon optimization: Adjusting the coding sequence to match yeast codon preferences can significantly enhance expression levels without altering the amino acid sequence.

• Fermentation parameters optimization:

  • Culture medium composition

  • Temperature control

  • Induction timing and inducer concentration

  • pH maintenance

  • Oxygenation levels

• Solubility enhancement tags: For variants prone to aggregation, solubility-enhancing fusion tags can be employed, though these require subsequent removal.

• Refolding protocol refinements: For proteins expressed as inclusion bodies, optimizing the refolding buffer composition is crucial. Including 1M arginine, maintaining appropriate pH (8.0), adding EDTA (5 mM), and controlling temperature (4°C) significantly improves refolding efficiency .

• Two-stage purification: Implementing a multi-step purification approach combining affinity chromatography with ion exchange and/or size exclusion chromatography can improve both yield and purity .

Through these approaches, researchers have achieved expression levels of 0.6-0.8 μg/ml in culture medium and final purified yields of 0.3-0.4 mg per liter of culture with recovery rates around 51% .

What are the appropriate biological assays for confirming the activity of yeast-expressed human IL-4?

Multiple complementary biological assays are essential for comprehensive activity assessment of yeast-expressed human IL-4:

• B-cell co-stimulator assays: Evaluating IL-4's ability to enhance B-cell proliferation in the presence of anti-IgM antibodies confirms a key biological function .

• T-cell proliferation assays: Measuring IL-4-induced proliferation of responsive T-cell lines verifies activity on another key target cell type .

• CD23 induction: Quantifying the upregulation of CD23 (the low-affinity receptor for IgE) expression on tonsillar B-cells provides a sensitive functional readout. Half-maximal biological activity of properly folded recombinant IL-4 is typically achieved at concentrations around 120 pM .

• TF-1 cell proliferation: This human erythroleukemic cell line proliferates in response to IL-4 and provides a quantitative dose-response relationship. This assay is particularly useful for determining inhibitory constants (Ki) when evaluating IL-4 antagonists .

• HEK-Blue SEAP reporter assays: These engineered cell lines produce secreted embryonic alkaline phosphatase (SEAP) in response to IL-4 signaling, offering a high-throughput colorimetric readout .

• Receptor binding studies: While not directly measuring function, receptor binding assays using 125I-labeled IL-4 can confirm proper interaction with IL-4 receptors, showing characteristic high-affinity binding (Kd = 100 pM) .

These assays collectively provide a comprehensive activity profile and can reveal subtle differences between IL-4 variants or expression systems.

How does yeast-expressed IL-4 compare functionally to IL-4 produced in other expression systems?

Functional comparison of IL-4 from different expression systems reveals important differences:

• Receptor binding characteristics:

  • Yeast-expressed IL-4 demonstrates high-affinity binding to IL-4Rα comparable to mammalian-expressed versions, with properly folded recombinant IL-4 showing Kd values around 100 pM .

  • Engineered variants with the F82D mutation can show enhanced receptor affinity regardless of expression system .

• Glycosylation differences:

  • Yeast-derived IL-4 typically contains high-mannose type glycans lacking terminal sialic acids.

  • Mammalian cell-produced IL-4 contains complex or hybrid N-glycans with terminal sialic acid residues, which affects properties like proteolytic stability .

  • Specifically, yeast-derived IL-4 with a single large N-glycan (~20 kDa) shows reduced proteolytic stability (half-life of 2.6 hours) compared to mammalian cell-derived IL-4 with multiple smaller N-glycans .

• Functional activity comparison:

  Expression System	Proteolytic Half-life	Receptor Affinity	Glycosylation Pattern
  Yeast (P. pastoris)	2.6 hours	Similar to mammalian	Single large poly-mannose (~20 kDa)
  HEK293 cells (single N-glycan)	Similar to yeast	Similar to yeast	Smaller complex/hybrid (~2-3 kDa)
  HEK293 cells (multiple N-glycans)	10.5-25 hours	Similar to yeast	Multiple complex/hybrid
  E. coli	Lowest	Similar	None

• Structure-function relationships:

  • The critical disulfide bonds form correctly in yeast systems, maintaining the tertiary structure essential for function .

  • Thermospray liquid chromatography-mass spectrometry confirms that yeast-produced IL-4 has the correct N-terminus and C-terminal glycosylation pattern .

These comparisons highlight that while core functionality is preserved across expression systems, glycosylation differences significantly impact pharmacokinetic properties like proteolytic stability .

How can researchers develop and characterize IL-4 mimetics using yeast expression platforms?

Developing IL-4 mimetics using yeast expression platforms involves several sophisticated approaches:

• De novo design strategy:

  • Beginning with an engineered scaffold (such as an IL-2 mimetic scaffold) that provides structural stability .

  • Computationally designing the mimetic by identifying key binding residues from IL-4 that interact with receptor components .

  • Grafting these critical binding residues onto the stable scaffold to create a preliminary mimetic design .

• Directed evolution in yeast:

  • Subjecting the initial design to random mutagenesis to generate variant libraries .

  • Using yeast surface display systems to enrich for variants with enhanced receptor binding .

  • Selecting clones with highest affinity for receptor components (e.g., mIL-4Rα) .

• Expression and purification:

  • Expressing selected clones in appropriate expression systems (yeast or E. coli) .

  • Purifying using established protocols adapted for the specific construct design .

• Comprehensive characterization:

  • Biophysical characterization: Evaluating stability, comparing binding affinities between mimetics and natural IL-4 for receptor components .

  • Functional assessment: Testing signaling profiles to determine receptor complex specificity (e.g., type I vs. type II IL-4 receptor complexes) .

  • In vivo validation: Confirming that the mimetics recapitulate physiological functions of IL-4 in cellular and animal models .

• Application development:

  • Exploring incorporation into biomaterials: Due to their hyperstability, well-designed mimetics can be incorporated into sophisticated biomaterials, including three-dimensional-printed scaffolds that require heat processing .

This approach has successfully yielded IL-4 cytokine mimetics (Neo-4) that signal exclusively through the type I IL-4 receptor complex while exhibiting enhanced stability compared to natural IL-4 .

How can IL-4 antagonists expressed in yeast systems be applied in studying type 2 inflammation mechanisms?

IL-4 antagonists expressed in yeast systems offer powerful tools for dissecting type 2 inflammation mechanisms:

• Investigating IL-4's paradoxical roles: IL-4 both suppresses type 1 inflammation and mediates type 2 inflammation through a positive feedback loop in TH2 cells. Antagonists can help delineate these dual functions by selectively blocking specific pathways .

• Studying allergic disease pathogenesis: IL-4 antagonists can be used to investigate how IL-4 drives allergic responses by:

  • Blocking IL-4-driven immunoglobulin class switching in B cells

  • Inhibiting IL-4-mediated eosinophil transmigration

  • Preventing increased mucus secretion

• Dissecting receptor-specific signaling: Engineered antagonists that selectively block either type I or type II receptor complex formation (like Neo-4 that signals exclusively through type I receptor) can elucidate the distinct contributions of each signaling pathway to inflammation .

• Investigating host-pathogen interactions: IL-4 impairs host defense against intracellular pathogens, including fungi like Histoplasma capsulatum. IL-4 antagonists can help elucidate the mechanisms behind this immunosuppression .

• Time-course studies: Using antagonists to block IL-4 at different stages of inflammatory response can reveal critical windows where IL-4 signaling drives disease progression, particularly in the early phase (around day 3) of infection models .

These applications leverage yeast-expressed antagonists as precision tools for mechanistic studies, owing to their high specificity, tunable properties, and capacity for modification.

What considerations are important when designing IL-4 glycovariants for extended half-life and reduced immunogenicity?

Designing IL-4 glycovariants with extended half-life and reduced immunogenicity requires strategic considerations:

• N-glycosylation site selection:

  • Sites must be outside of secondary structure elements to ensure recognition by glycosyltransferases in the endoplasmic reticulum .

  • Positions should not interfere with IL-4-IL-4Rα binding to maintain antagonist efficacy .

  • Key validated positions include Q20N, T28N, and K61N, which can be combined in a multi-glycosylated variant .

• Glycan architecture and composition:

  • Mammalian cell-derived complex N-glycans with terminal sialic acid residues provide superior proteolytic stability compared to yeast-derived poly-mannose glycans .

  • Proteolytic stability data demonstrates this difference clearly:

  IL-4 Variant Source	Glycosylation Type	Proteolytic Half-life
  Multi-glycosylated HEK293	Multiple complex N-glycans	25 hours
  Single-glycosylated HEK293	Single complex N-glycan	10.5 hours
  P. pastoris (yeast)	Single large poly-mannose glycan (~20 kDa)	2.6 hours

• Chemical vs. biosynthetic glycoengineering:

  • Chemical glycosylation offers precise control over glycan structure and position but may introduce non-natural linkages .

  • Biosynthetic approaches using appropriate expression hosts produce naturally-linked glycans but with less structural control .

  • A combined approach may offer optimal results: introducing multiple N-glycosylation sites biosynthetically while using chemical glycosylation at a specific position (e.g., R121C) to create antagonist functionality .

• Alternative to PEGylation:

  • Glycoengineering provides advantages over PEGylation, which can be heterogeneous, affect antagonist efficacy, and show poor degradability leading to liver accumulation .

  • N-glycans are naturally present on many secreted proteins, reducing immunogenicity concerns compared to synthetic polymers like PEG .

These considerations enable researchers to design IL-4 glycovariants with significantly improved pharmacokinetic properties while maintaining desired functional characteristics.

What are the emerging applications of hyperstable IL-4 mimetics in biomaterial development and tissue engineering?

Hyperstable IL-4 mimetics offer groundbreaking opportunities in biomaterial development and tissue engineering:

• Integration with heat-processed biomaterials:

  • Conventional cytokines typically denature during biomaterial processing that involves heating.

  • Computationally designed IL-4 mimetics (Neo-4) demonstrate exceptional thermal stability, allowing direct incorporation into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds .

• Controlled immunomodulation in tissue engineering:

  • IL-4 promotes type 2 immune responses that can support wound healing and tissue remodeling.

  • Stable mimetics incorporated into scaffolds can provide sustained immunomodulatory signals to influence tissue repair and regeneration .

• Cell-type-specific targeting:

  • Engineered mimetics that signal exclusively through the type I IL-4 receptor complex allow targeting of specific cell populations (those expressing the complete type I receptor) .

  • This selective activity enables more precise control over cellular responses in complex engineered tissues .

• Long-term stability in implantable materials:

  • Hyperstable mimetics maintain activity in implantable materials over extended periods, potentially eliminating the need for repeated administration.

  • This sustained activity is crucial for applications requiring prolonged immunomodulation, such as implants for chronic inflammatory conditions .

• Investigation of differential IL-4 signaling:

  • Materials incorporating mimetics that selectively activate type I receptors provide previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors .

  • These materials become valuable research tools for studying receptor-specific effects in tissue microenvironments .

These applications represent a significant advance over conventional approaches using natural cytokines, opening new possibilities for precision immunomodulation in regenerative medicine and tissue engineering.

What strategies can address purity and heterogeneity issues in yeast-expressed IL-4 preparations?

Addressing purity and heterogeneity challenges in yeast-expressed IL-4 requires systematic approaches:

• Multi-step purification strategy:

  • Initial affinity chromatography using concanavalin A-Sepharose to capture glycoproteins .

  • Intermediate ion-exchange chromatography using S-Sepharose Fast Flow to separate based on charge characteristics .

  • Final polishing step with C18 reverse-phase HPLC to achieve high purity .

  • This comprehensive approach can yield highly purified IL-4 with recovery rates around 51% .

• Managing glycoform heterogeneity:

  • Introducing specific N-glycosylation sites at well-defined positions (Q20N, T28N, K61N) to control location and number of glycans .

  • Enzymatic processing with specific glycosidases (like PNGase F) can be used to remove or homogenize N-linked oligosaccharides .

  • Using modified yeast strains with altered glycosylation pathways can produce more uniform glycan structures.

• Addressing proteolytic degradation:

  • Incorporating protease inhibitors during purification.

  • Engineering increased proteolytic stability through strategic glycosylation - HEK293 cell-derived IL-4 with multiple N-glycans shows significantly improved proteolytic half-life (25 hours) compared to yeast-derived variants (2.6 hours) .

• Analytical characterization:

  • Comprehensive mass spectrometry to confirm protein identity, glycosylation status, and detect potential modifications .

  • SDS-PAGE analysis to assess purity and molecular weight distribution .

  • Thiol titration to verify correct disulfide bond formation and absence of free cysteine residues .

  • Functional assays to confirm biological activity and detect inactive species .

These approaches collectively address the major sources of heterogeneity in yeast-expressed IL-4 preparations, enabling production of consistent, high-quality material for research applications.

How can researchers troubleshoot expression and folding issues when producing IL-4 variants in yeast?

Troubleshooting expression and folding issues for IL-4 variants in yeast requires systematic investigation of several critical parameters:

• Diagnosing low expression yields:

  • Verify vector construction and sequence integrity

  • Optimize codon usage for yeast preference

  • Evaluate secretion signal efficiency (prepro alpha-mating factor is effective for IL-4)

  • Adjust induction conditions (timing, temperature, media composition)

  • Consider alternative yeast strains with different secretory capacities

• Addressing protein misfolding:

  • Optimize refolding buffer composition: 1M arginine significantly enhances folding efficiency

  • Adjust redox conditions: The correct ratio of oxidized to reduced glutathione is critical for proper disulfide formation

  • Control protein concentration: Typically 1-1.5 mg/mL for bacterial-derived insoluble protein or 1 mg/mL for HEK293 cell-derived protein

  • Modify refolding temperature: 4°C for 72 hours often yields optimal results

  • For variants with introduced cysteines, address potential mixed disulfide formation with glutathione

• Resolving disulfide bond issues:

  • Verify correct formation of all three native disulfide bonds using thiol titration

  • For variants with additional cysteines (e.g., R121C), enzymatic deglutathionylation may be necessary

  • When chemical reduction is required, carefully optimize conditions to avoid disrupting native disulfide bonds

• Evaluation and verification:

  • Analytical techniques: Mass spectrometry to confirm protein integrity and modifications

  • Functional assays: Multiple bioassays to verify activity (B-cell co-stimulation, T-cell proliferation, CD23 induction)

  • Receptor binding: Confirm proper interaction with IL-4 receptors using labeled IL-4 in binding studies

For IL-4 variants designed as antagonists, additional considerations include verifying that modifications achieve the desired antagonist function without compromising IL-4Rα binding, which can be assessed using inhibition constants (Ki) in functional assays .

What methods can researchers use to verify the specific binding of IL-4 variants to different receptor complexes?

Verifying specific binding of IL-4 variants to different receptor complexes requires sophisticated methods that can distinguish between type I (IL-4Rα/γc) and type II (IL-4Rα/IL-13Rα1) receptor interactions:

• Surface Plasmon Resonance (SPR) analysis:

  • Sequential binding studies using immobilized receptor components (IL-4Rα, γc, IL-13Rα1)

  • Determination of binding kinetics (kon, koff) and affinity constants (KD) for individual receptor components

  • Analysis of heterotrimeric complex formation through sequential receptor addition

  • This approach can identify variants like Neo-4 that signal exclusively through the type I IL-4 receptor complex

• Cell-based receptor specificity assays:

  • Using cell lines expressing either type I or type II receptor complexes exclusively

  • Comparing signaling responses (e.g., STAT6 phosphorylation) in each cell type

  • Competitive binding studies with labeled wild-type IL-4 to determine receptor preference

  • These assays can reveal differential activation patterns between receptor complexes

• Receptor-ligand cross-linking studies:

  • Chemical cross-linking of labeled IL-4 variants to cell-surface receptors

  • SDS-PAGE analysis to visualize receptor complexes

  • Comparison of cross-linked complex patterns between wild-type and variant IL-4

  • This technique has successfully identified the IL-4 receptor as a 124 kDa cell-surface protein

• Functional signaling pathway analysis:

  • Phospho-flow cytometry to measure activation of downstream signaling molecules

  • Reporter gene assays using pathway-specific reporters

  • Transcriptional profiling to identify receptor-specific gene expression signatures

  • These approaches can distinguish subtle differences in signaling quality between variants

• Competitive binding assays:

  • Using 125I-labeled IL-4 in equilibrium binding studies

  • Determining displacement patterns by unlabeled variants

  • Analysis of binding to specific cell types expressing different receptor components

  • This method has successfully characterized high-affinity receptor binding (Kd = 100 pM) and quantified receptor numbers (1100 receptors per cell on Raji B-cells)

These complementary approaches provide comprehensive characterization of how IL-4 variants interact with different receptor complexes, essential information for designing cytokine mimetics or antagonists with specific targeting properties.