Recombinant Human Transforming Growth Factor-beta (rhTGF-beta) Engineering Service

Recombinant Human Transforming Growth Factor-beta (rhTGF-beta) is a pleiotropic cytokine crucial in biomedicine for regulating cell proliferation, differentiation, and tissue repair. TGF-beta is naturally a secreted, dimeric protein stabilized by disulfide bonds. Industrial production in Escherichia coli (E. coli) is a common choice for high expression, but faces two major difficulties: Low expression when trying to achieve a soluble, active form, and the product is prone to aggregation during the necessary refolding process after isolation from inclusion bodies.

CD Biosynsis offers an integrated protein and process engineering service to produce high-quality rhTGF-beta. The challenge of low expression and poor solubility is addressed by Modification of fusion expression system in Escherichia coli . We replace the TGF-beta pre-pro peptide with an optimized, highly soluble fusion tag (e.g., Trx, GST, or Maltose-binding protein (MBP)) that increases the accumulation of the rhTGF-beta fusion protein within E. coli. To tackle the critical quality issue ( prone to aggregation ), we focus on Optimization of protein refolding process . We screen and optimize a panel of refolding conditions, including the use of redox shuttles (e.g., GSH/GSSG ratio), osmolytes, and chaperone-like additives (e.g., L-Arginine), to maximize the yield of correctly folded and dimeric rhTGF-beta while minimizing aggregation. This combined strategy ensures a high yield of soluble protein precursor and its efficient conversion to the active dimer form.

Pain Points

Achieving cost-effective, active rhTGF-beta production faces these key challenges in E. coli:

• Low Expression (Active Form): While total protein expression is high, the native rhTGF-beta without a suitable tag often forms insoluble inclusion bodies or is rapidly degraded, leading to low expression of the functional product.
• Prone to Aggregation: TGF-beta requires the correct formation of disulfide bonds and dimerization. During the refolding process , the exposed hydrophobic regions often lead to competing intermolecular interactions and aggregation , drastically reducing yield.
• Inefficient Disulfide Bond Formation: Correct folding requires multiple disulfide bonds. Achieving the native bond pattern (essential for activity) is difficult during in vitro refolding.
• Fusion Tag Cleavage: The fusion protein requires efficient and complete cleavage to yield the final, active rhTGF-beta, which can itself be a source of product loss and purification complexity.

A successful solution must ensure high yield of soluble precursor and its efficient conversion to the native active dimer.

Solutions

CD Biosynsis utilizes advanced protein and process engineering to optimize rhTGF-beta production:

Modification of Fusion Expression System in E. coli

           

We screen and optimize highly soluble fusion tags (e.g., MBP, NusA) to maximize the soluble precursor yield, addressing low expression .

Optimization of Protein Refolding Process

We optimize buffer composition, redox conditions (GSH/GSSG), and additives (Arginine) to prevent aggregation and maximize active dimer formation.

Soluble Expression Strategies

We use E. coli strains optimized for disulfide bond formation (e.g., trxB gor mutants) to promote active folding directly within the cytoplasm.

Tag Cleavage and Purification Optimization

We select a fusion tag and protease cleavage site (e.g., TEV) to ensure high cleavage yield and easy tag removal during downstream processing.

This systematic approach targets maximum soluble precursor yield followed by efficient conversion to the active dimeric cytokine.

Advantages

Our rhTGF-beta engineering service is dedicated to pursuing the following production goals:

High Soluble Precursor Yield

Optimized fusion system prevents inclusion body formation, resulting in high expression of the necessary rhTGF-beta precursor.

Maximal Active Dimer Yield

Refolding optimization minimizes non-specific interactions, significantly reducing the fraction prone to aggregation and maximizing the active dimer recovery.

Biologically Active Product Icon

Precise control over disulfide bond formation ensures the product is structurally native and possesses full biological activity.

Cost-Effective E. coli Platform Icon

Using the high-density and low-cost E. coli system for production maintains economic competitiveness.

Consistent Quality and Purity

Optimized refolding ensures minimal presence of misfolded, aggregated side products in the final material.

We provide a competitive, high-quality manufacturing solution for rhTGF-beta production.

Process

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

• Fusion Vector Design: Clone the TGF-beta gene with a selected high-solubility fusion tag (e.g., MBP or GST) and protease cleavage site (e.g., TEV) into an E. coli expression plasmid.
• Expression Optimization: Optimize induction conditions (inducer concentration, temperature) to maximize soluble fusion protein yield.
• Inclusion Body Solubilization: Develop a protocol for the efficient solubilization of the inclusion body fraction using denaturants (e.g., urea, guanidine HCl).
• Refolding Screening: High-throughput screen refolding buffers for optimal concentrations of denaturants, redox shuttles (GSH/GSSG), and aggregation suppressors (Arginine).
• Cleavage and Purification: Perform optimized protease cleavage and multi-step chromatography to isolate the final active rhTGF-beta dimer from the fusion tag and misfolded products.
• Functional Assay Validation: Perform a cell-based reporter gene assay (e.g., PAI-1 promoter induction) to confirm full biological activity (EC_50) of the final product.

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

Explore the potential for a high-quality, high-yield rhTGF-beta supply. CD Biosynsis provides customized protein expression and refolding solutions:

• Detailed Refolding Yield Report , showing the conversion rate of denatured protein to active, soluble dimer after the optimized refolding process.
• Consultation on fermentation scale-up of the E. coli host strain for industrial production.
• Experimental reports include complete raw data on biological EC_50 (Activity) and final product aggregation level (SEC-HPLC) , essential for quality control.