Recombinant Human Epidermal Growth Factor (rhEGF) Engineering Service

Recombinant Human Epidermal Growth Factor ( rhEGF), widely used in regenerative medicine and cosmetology, is a small protein stabilized by three disulfide bonds. Standard production in Escherichia coli cytoplasm poses two major challenges: prokaryotic expression is prone to degradation , as the small size and the presence of exposed sites make rhEGF a target for E. coli proteases. More critically, E. coli cytoplasm is a reducing environment, which hinders the formation of the three correct disulfide bonds, leading to low activity of the final product and requiring complex and costly in vitro refolding. Biosynthesis optimization can solve both folding and stability issues.

CD Biosynsis offers a synthetic biology service focused on engineering an active, stable rhEGF production system. Our core strategy involves modification of Escherichia coli secretion expression system . We utilize a signal peptide (e.g., PelB) to direct the rhEGF precursor into the E. coli periplasm, which is an oxidizing environment. This ensures the correct, spontaneous formation of the three disulfide bonds, yielding a properly folded and highly active protein. Secretion also protects the product from cytoplasmic proteases. This is coupled with mutation of protein stability . We employ site-directed mutagenesis to introduce specific point mutations (e.g., substitution of Ala with Cys at non-disulfide sites for enhanced dimerization, or substitution of protease-sensitive residues) that are known to increase rhEGF's thermal and chemical stability without compromising receptor binding. This integrated approach aims to deliver high yields of a biologically active, stable, and correctly folded rhEGF directly into the periplasm or medium, significantly streamlining purification and enhancing clinical utility.

Pain Points

Efficient and stable rhEGF production faces these key challenges:

• Prokaryotic Expression is Prone to Degradation: The small size of rhEGF (6 kDa) makes it highly susceptible to E. coli cytoplasmic and periplasmic proteases ( Lon, OmpT), resulting in significant product loss.
• Low Activity: Incorrect folding due to the reducing environment of the E. coli cytoplasm leads to improper formation of the three native disulfide bonds, yielding inactive inclusion bodies or misfolded monomers.
• Refolding Complexity and Cost: Purification from inclusion bodies requires time-consuming, expensive, and low-yield in vitro refolding protocols to restore biological activity.
• Low Thermal and pH Stability: The native protein has limited stability under manufacturing conditions and during long-term storage, hindering formulation.

A successful solution must ensure correct in vivo folding while simultaneously increasing the intrinsic stability of the protein molecule.

Solutions

CD Biosynsis utilizes advanced expression system and protein engineering to optimize active, stable rhEGF production in E. coli:

Modification of E. coli Secretion Expression System

           

We employ a robust periplasmic secretion system (using PelB or DsbA signal sequences) to transfer rhEGF to the oxidizing periplasm, enabling spontaneous and correct disulfide bond formation .

Mutation of Protein Stability

We use site-directed mutagenesis to modify amino acid residues that are sensitive to proteolysis or that contribute to thermal instability, creating protease-resistant, stable variants without loss of function.

Protease-Deficient Host Strain Selection

We screen and utilize specialized E. coli strains (e.g., BL21 series with lon and ompT deletions) that have reduced protease activity , minimizing product degradation during and after expression.

Fusion Tag-Mediated Solubility

If periplasmic expression yields are low, a highly soluble N-terminal fusion tag (e.g., MBP or Trx) can be used to promote solubility and protect the protein from degradation.

This systematic approach generates a correctly folded, active, and stable product directly in a production-friendly environment.

Advantages

Our rhEGF engineering service is dedicated to pursuing the following production goals:

High Biological Activity

Periplasmic secretion ensures the correct formation of disulfide bonds in vivo, maximizing the active fraction of the protein.

Enhanced Product Stability

Mutation of stability-limiting sites improves resistance to heat and proteases , reducing degradation during manufacture and storage.

Simplified Downstream Processing

Secreted active protein eliminates the complex refolding step and significantly lowers the cost of goods (COG). [Image of Cost Reduction Icon]

Increased Final Titer

Minimizing degradation via protease-deficient strains and protection in the periplasm maximizes the recoverable yield .

High Purity for Clinical Use

Periplasmic extracts are generally cleaner than whole-cell lysates, facilitating the production of pharmaceutical-grade rhEGF .

We provide a reliable and cost-effective platform for active rhEGF biomanufacturing.

Process

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

• Gene Design and Signal Peptide Fusion: Design the rhEGF gene with codon optimization and fuse it to a periplasmic signal peptide ( PelB) for secretion.
• Protein Stability Mutagenesis: Introduce stability-enhancing point mutations based on structural analysis and known protease cleavage sites.
• Expression Host Selection: Select and transform the expression vector into a protease-deficient E. coli host strain (e.g., BL21 (DE3) $\Delta lon \Delta ompT$ derivative).
• Expression and Purification: Optimize induction conditions (e.g., IPTG concentration, temperature) and perform periplasmic extraction followed by chromatography.
• Functional and Purity Assays: Verify the product's integrity via RP-HPLC and confirm biological activity using a cell proliferation assay (e.g., NRK-49F cell line). Confirm disulfide bond formation via Ellman's assay.
• Result Report Output: Compile a detailed Experimental Report including gene modification data, purification protocols, and functional data (activity units/mg, stability profile) , supporting regulatory filing.

Technical communication is maintained throughout the process, focusing on timely feedback regarding folding efficiency and activity retention.

Explore the potential for a stable, highly active rhEGF therapeutic. CD Biosynsis provides customized protein engineering solutions:

• Detailed Folding Efficiency and Stability Report , demonstrating the improved activity and half-life of the engineered variant.
• Consultation on optimized E. coli fermentation and periplasmic extraction protocols for high-yield recovery.
• Experimental reports include complete raw data on biological activity ( IU/mg) and final product yield ( mg/L) of the active monomer , essential for cGMP readiness.