Recombinant Spider Silk Protein RSpS Engineering Service

Recombinant Spider Silk Protein RSpS is sought after for high-performance Textiles/Biomaterials e.g. surgical sutures, lightweight composites. Challenges in microbial production include: The repetitive and large protein structure leads to low solubility and tendency to form insoluble inclusion bodies during expression. This, along with expression size and toxicity to the host, results in low final yield of spinnable protein . A further challenge is the difficulty in replicating the complex mechanical properties of native spider silk fibers in synthetic processes.

CD Biosynsis offers a tailored protein and host engineering strategy for efficient RSpS production. Our approach centers on: Host Modification: Utilize methylotrophic yeast Pichia pastoris for high-density, high-yield secretion . Pichia can handle the large, repetitive protein size better than E. coli and facilitates secretion, reducing inclusion body formation. We enhance purification efficiency using Fusion Tags: Use Elastin-like Peptides ELPs or Histidine tags to improve solubility and facilitate non-chromatographic purification . ELPs allow for efficient, low-cost Inverse Transition Cycling ITC purification. Finally, we ensure optimal expression and function with Artificial Gene Design: Optimize the gene sequence codon optimization, repetitive motif control for high expression and subsequent fiber assembly .

Pain Points

Industrial RSpS production faces these key challenges in biomanufacturing:

• Inclusion Body Formation: Due to the highly repetitive and hydrophobic nature of spider silk proteins, they often misfold and aggregate as insoluble inclusion bodies in the cytosol of hosts like E. coli.
• Low Spinnable Yield: Inclusion body formation requires costly and harsh refolding steps. Also, the large protein size up to 300 kDa and its metabolic burden or toxicity to the host results in low final protein titer .
• Replicating Mechanical Properties: Synthetic processes often fail to reproduce the microstructure of native silk, resulting in fibers with inferior strength and toughness compared to natural silk.
• Purification Complexity: Separating and purifying the soluble protein or refolded protein from the broth is often dependent on expensive multi-step chromatography e.g. IMAC .

A high-value RSpS product requires high-yield soluble expression and efficient low-cost purification.

Solutions

CD Biosynsis utilizes advanced protein and host engineering to optimize RSpS production:

Host Switching Pichia pastoris

           

We use methylotrophic yeast Pichia pastoris as a host. Its strong AOX1 promoter and ability to secrete soluble protein reducing I.B. formation are ideal for large RSpS monomers.

Fusion Tag Purification ELPs

We engineer the RSpS with thermo-responsive Elastin-like Peptides ELPs to enable cost-effective non-chromatographic purification via Inverse Transition Cycling ITC.

Artificial Gene Design Codon Optimization

We optimize the synthetic gene sequence to match the tRNA pool of Pichia pastoris and design the repetitive motifs for maximal protein yield and subsequent self-assembly into fibers.

Tailored Spinnable RSpS Monomer Design

We design the monomer construct to include critical domains e.g. terminal domains, amorphous spacers that drive self-assembly and improve mechanical properties during fiber spinning. [Image of High Conversion Efficiency Icon]

This integrated strategy addresses expression, solubility, purification, and final material properties.

Advantages

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

High Soluble Protein Yield Icon

Pichia host and secretion strategy significantly reduces insoluble inclusion body formation , maximizing recoverable protein.

Reduced Downstream Purification Cost Icon

ELP fusion tags enable efficient, low-cost non-chromatographic purification ITC compared to traditional columns. [Image of Cost Reduction Icon]

Enhanced Mechanical Properties Icon

Optimized gene design ensures the final protein monomer retains the necessary self-assembly and crosslinking motifs .

Optimal Codon Usage for Expression Icon

Codon optimization for the Pichia host facilitates efficient translation of the large, repetitive RSpS gene.

Scalable Production Platform Icon

High-density fermentation in Pichia and non-chromatographic purification support industrial scale-up .

We provide RSpS tailored for optimal spinnability and performance in final material applications.

Process

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

• Gene Synthesis and Design: Synthesize the RSpS gene with codon optimization for Pichia pastoris and incorporate a secretion signal e.g. alpha factor and an ELP fusion tag.
• Host Transformation and Screening: Integrate the optimized gene into the Pichia genome and screen for high copy number clones that exhibit the highest secretion titer of soluble protein .
• Fermentation Optimization: Optimize induction conditions e.g. methanol feed rate, pH, temperature in high-density fed-batch fermentation to maximize RSpS yield.
• Purification and Characterization: Purify the secreted protein using ELP-based Inverse Transition Cycling and verify solubility and purity e.g. SDS-PAGE, SEC-HPLC.
• Material Assembly Validation: Provide consultation on wet-spinning or microfluidic fiber assembly protocols and validate the mechanical properties of the final fiber.

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

Explore the potential for a high-performance, cost-effective RSpS supply. CD Biosynsis provides customized protein and process engineering solutions:

• Detailed Soluble RSpS Titer Report g/L from high-density fermentation and purification yield data.
• Consultation on ELP purification protocols and tag removal strategies, including protease cleavage sites.
• Experimental reports include complete raw data on protein purity, monomer integrity, and characterization e.g. DLS or SEC , crucial for fiber spinning.