Recombinant Human Interleukin-4 (rhIL- 4) Engineering Service

Recombinant Human Interleukin-4 (rhIL- 4) is a critical cytokine used in biomedicine to regulate immune responses, particularly Th2 cell differentiation and IgE production. IL- 4 requires correct folding and a specific tertiary structure (stabilized by disulfide bonds) for activity. Production in prokaryotic expression systems (E. coli) is highly desirable for cost efficiency but often results in the product having No activity in prokaryotic expression . This is because E. coli's cytoplasm lacks the necessary machinery (chaperones, oxidizing environment) to form the correct disulfide bonds. Furthermore, once produced, the protein is prone to degradation by bacterial proteases, leading to low yield and poor purity.

CD Biosynsis offers an integrated protein and host engineering service for high-quality rhIL- 4 production. The challenge of achieving active product ( no activity in prokaryotic expression ) is addressed by Modification of Escherichia coli secretion expression system . We utilize E. coli strains engineered for the oxidizing periplasm (e.g., Origami or SHuffle strains) and employ specific secretion tags (e.g., DsbA or PelB leader sequence) to channel the rhIL- 4 into the periplasmic space. This oxidizing environment facilitates the correct formation of the two critical disulfide bonds necessary for biological activity. To tackle the stability issue ( prone to degradation ), we focus on Mutation of protein stability . We use rational design (sequence analysis or molecular dynamics) or directed evolution to introduce specific point mutations (e.g., replacing protease cleavage sites or introducing stabilizing residues) to enhance the intrinsic structural stability of rhIL- 4 against both thermal stress and bacterial proteases. This combined strategy ensures that the rhIL- 4 is correctly folded for activity and protected against degradation for high yield.

Pain Points

Achieving stable, high-activity rhIL- 4 production in E. coli faces these key challenges:

• No Activity in Prokaryotic Expression: When expressed in the E. coli cytoplasm, the rhIL- 4 cannot form its two essential disulfide bonds due to the reducing environment, resulting in a misfolded, biologically inactive product .
• Prone to Degradation: IL- 4 is a small, relatively unstable protein that is highly susceptible to proteolytic cleavage by native E. coli proteases (e.g., Lon, OmpT) during expression and recovery, leading to low functional yield .
• Inefficient Secretion or Translocation: Moving the protein to the oxidizing periplasm is critical, but the use of generic or non-optimized secretion tags can lead to poor efficiency and aggregation in the inner membrane.
• Difficult Refolding Alternative: IL- 4 can be produced in inclusion bodies and refolded, but the in vitro refolding process is complex, low-yield, and also prone to aggregation .

A successful solution must ensure correct folding occurs in vivo and protect the product from degradation.

Solutions

CD Biosynsis utilizes advanced protein and host engineering to optimize rhIL- 4 production:

Modification of E. coli Secretion Expression System

           

We employ periplasmic expression in disulfide-bond-competent strains (e.g., SHuffle B strain) with an optimized PelB leader sequence, solving the no activity issue.

Mutation of Protein Stability

We introduce site-specific mutations to eliminate known protease cleavage sites and increase the thermal stability of rhIL- 4, mitigating the prone to degradation challenge.

Host Protease Knockout

We use E. coli expression strains with multiple protease gene deletions (e.g., ΔompT, Δlon) to minimize extracellular and intracellular protein hydrolysis.

Disulfide Bond Isomerization Assistance

The periplasmic space is further engineered to co-express DsbC to enhance the correct isomeric formation of the rhIL- 4 disulfide bonds.

This systematic approach overcomes the challenges of correct folding and protein stability in E. coli production.

Advantages

Our rhIL- 4 engineering service is dedicated to pursuing the following production goals:

High Biological Activity

Periplasmic expression in SHuffle strains ensures correct disulfide bond formation , solving the no activity in prokaryotic expression issue.

Enhanced Product Stability

Mutations and protease knockout strains minimize proteolytic degradation, making the product less prone to degradation during storage and purification.

High Soluble Yield Icon

Combining high expression, efficient secretion, and stability engineering results in a high yield of the desired active product.

Simplified Purification Icon

Periplasmic expression simplifies initial recovery by using osmotic shock, bypassing complex refolding steps.

Cost-Effective Production Icon

The cost advantages of the E. coli platform are fully realized due to the high quality and yield of the active product.

We provide a cost-effective, high-quality, and highly active platform for rhIL- 4 production.

Process

Our rhIL- 4 engineering service follows a rigorous, multi-stage research workflow:

• rhIL- 4 Gene Design: Codon optimize the IL- 4 gene and introduce an optimized PelB secretion leader sequence .
• Stability Engineering: Introduce site-specific mutations based on protease cleavage site prediction and stability analysis to generate a robust IL- 4 variant.
• Expression System Selection: Clone the modified gene into a high-copy vector and transform it into a disulfide-bond-competent E. coli strain (e.g., SHuffle T7).
• Secretion Optimization: Optimize induction conditions (temperature, inducer concentration) to maximize translocation to the periplasm while minimizing protein stress and degradation.
• Quality and Purity Assessment: Use Western Blot and HPLC to confirm minimal degradation and high purity.
• Functional Assay Validation: Perform a cell-based proliferation or STAT6 phosphorylation assay to confirm full biological activity (EC}_{50) of the final product, directly addressing the initial no activity challenge.

Technical communication is maintained throughout the process, focusing on timely feedback regarding yield and product quality attributes.

Explore the potential for a stable, high-activity rhIL- 4 supply. CD Biosynsis provides customized expression system and protein engineering solutions:

• Detailed Periplasmic Titer and Activity Report , showing the yield of the correctly folded rhIL- 4 variant.
• Consultation on fermentation process control (e.g., DO control for periplasmic expression) to maintain optimal folding conditions.
• Experimental reports include complete raw data on biological EC}_{50 and stability testing (e.g., half-life under various conditions) , demonstrating product robustness.