Recombinant Human Fibroblast Growth Factor (rhFGF) Engineering Service

Recombinant Human Fibroblast Growth Factor ( rhFGF), a critical signaling protein for cell proliferation, wound healing, and tissue regeneration, is widely used in pharmaceuticals and cosmetology. Production in microbial systems often faces two major hurdles: low expression level due to codon bias, complex folding requirements, or promoter strength issues; and prone to degradation by host-secreted proteases, especially when targeting a high-yield secretion system. Furthermore, FGFs are sensitive to shear stress and thermal instability. Optimization is crucial for achieving high yields of an active, stable protein.

CD Biosynsis offers a synthetic biology service focused on engineering a highly efficient rhFGF production system using Pichia pastoris , an established yeast platform known for high-level secretion and proper post-translational modifications. Our core strategy involves optimization of Pichia pastoris secretion system . We rigorously optimize the expression cassette, including the signal peptide ( e.g., alpha-factor or PIR), the strong AOX1 promoter, and gene copy number integration, to maximize the translocation and secretion rate of rhFGF. This is coupled with overexpression of protein folding chaperones . We co-express key Pichia chaperones and folding enzymes ( e.g., Kar2, PDI, ERO1) within the ER to enhance the protein folding capacity, reduce aggregation, and promote the correct formation of rhFGF's disulfide bonds. Additionally, we use protease-deficient strains and introduce specific protease cleavage site mutations to prevent degradation. This integrated approach aims to deliver a high-titer, correctly folded, biologically active rhFGF directly into the fermentation broth, simplifying downstream purification.

Pain Points

Efficient and stable rhFGF production faces these key challenges:

• Low Expression Level: Even with strong promoters, expression is often limited by codon usage incompatibility , poor mRNA stability, or saturation of the host's protein folding and secretion machinery.
• Prone to Degradation: Yeast cells secrete various proteases ( e.g., Yps1) into the medium. The secreted rhFGF is highly susceptible to these extracellular proteases , leading to fragmentation and loss of yield.
• Complex Folding Requirement: FGFs require precise disulfide bond formation and sometimes N-glycosylation for stability and function. Misfolding leads to the accumulation of inactive aggregates in the ER.
• Oligomerization and Aggregation: High concentration in the fermentation broth can cause non-specific aggregation or dimerization of rhFGF, reducing its monomeric activity.

A successful solution must simultaneously increase the expression capacity and protect the secreted product from degradation and aggregation.

Solutions

CD Biosynsis utilizes advanced protein and strain engineering to optimize active, stable rhFGF production in Pichia pastoris:

Optimization of Pichia pastoris Secretion System

           

We fine-tune the signal peptide, optimize DNA elements ( e.g., AOX1 promoter), and increase gene copy number integration to maximize secretion efficiency.

Overexpression of Protein Folding Chaperones

We co-express ER chaperones ( e.g., PDI, Kar2) to boost the folding capacity, ensuring rhFGF achieves its native conformation efficiently.

Protease Deletion and Site-Directed Mutagenesis

We use protease-deficient strains ( e.g., Delta yps1) and genetically modify rhFGF's surface residues to remove known protease cleavage sites , preventing degradation.

Codon Optimization and Fusion Partner Strategy

The rhFGF gene sequence is fully codon-optimized for Pichia and can be fused with a highly soluble, cleavable partner to enhance expression and stability.

This systematic approach increases the cell's productivity and protects the secreted protein, leading to a high-quality product.

Advantages

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

Dramatically Increased Expression Titer

Optimized promoter and multi-copy integration lead to significantly higher volumetric productivity than standard systems.

High Biological Activity

Enhanced chaperone assistance ensures correct disulfide bonding and native folding , maximizing the active fraction.

Reduced Product Degradation

Use of protease-deficient strains and site-directed mutagenesis protects the secreted rhFGF from proteolytic cleavage. [Image of Cost Reduction Icon]

Simplified Purification

Secretion into the medium means the product is separate from most host proteins, streamlining the initial recovery steps .

Scalable and Robust Host Icon

Pichia pastoris is highly suitable for large-scale fed-batch fermentation, providing a reliable platform for cGMP production.

We provide a reliable and efficient manufacturing platform for pharmaceutical-grade rhFGF.

Process

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

• Vector Construction: Assemble the optimized rhFGF gene ( codon-optimized and protease-site-mutated) with the alpha-factor signal peptide under the AOX1 promoter.
• Host Strain Transformation: Transform the construct into a protease-deficient Pichia strain ( e.g., KM71H derivative) and screen for high gene copy number integrants.
• Chaperone Co-expression: Co-integrate plasmids encoding PDI and Kar2 under constitutive or inducible promoters to boost the folding machinery capacity.
• Fermentation Optimization: Develop a high-cell-density fed-batch fermentation protocol tailored for the engineered strain, optimizing pH and methanol induction profiles.
• Functional and Purity Assays: Assess the product via SDS-PAGE and Western Blot to check for degradation. Confirm biological activity using a cell proliferation assay ( e.g., 3T3 cell line).
• Result Report Output: Compile a detailed Experimental Report including vector maps, gene copy number data, and fermentation metrics (active titer and degradation rate) , supporting commercial scale-up.

Technical communication is maintained throughout the process, focusing on timely feedback regarding folding yield and stability.

Explore the potential for a stable, high-yield rhFGF supply. CD Biosynsis provides customized protein engineering solutions:

• Detailed Secretion Efficiency and Stability Report , demonstrating the reduction in degradation products.
• Consultation on optimized primary recovery methods ( e.g., centrifugation and ultrafiltration) for the secreted protein broth.
• Experimental reports include complete raw data on biological activity ( U/mg) and total active protein yield ( mg/L) , essential for regulatory and manufacturing standards.