Recombinant Human Relaxin (rHRLX) Engineering Service

Recombinant Human Relaxin (rHRLX) is a two-chain polypeptide hormone linked by disulfide bonds, currently investigated for treating conditions like acute heart failure and fibrosis. Challenges in bioproduction stem from its complex structure: the A and B chains must be correctly processed and joined, resulting in low expression level in many hosts. Crucially, the mispairing of disulfide bonds affects activity severely, as rHRLX requires two inter-chain and one intra-chain disulfide bond to achieve its native, active conformation, making correct folding a major bottleneck.

CD Biosynsis offers a synthetic biology service focused on enhancing the yield and fidelity of rHRLX production. Our core strategy involves optimization of the Pichia pastoris secretion system (a highly efficient host for complex disulfide-bonded proteins) by optimizing signal peptides and protease cleavage sites. This is combined with overexpression of protein folding auxiliary factors (e.g., protein disulfide isomerase (PDI) and chaperones) to ensure the A and B chains are assembled and correctly folded within the endoplasmic reticulum (or secretory pathway). This integrated approach aims to deliver a high-yield, high-activity, and correctly folded rHRLX product.

Pain Points

Achieving cost-effective and high-activity rHRLX production faces these significant hurdles:

• Low Expression Level: Due to complex processing (leader sequence cleavage, chain joining), the overall yield of the mature, active rHRLX is often poor , hindering commercial scalability.
• Mispairing of Disulfide Bonds: rHRLX has a total of three disulfide bonds. Misfolding leads to incorrect bond formation , producing inactive aggregates or intermediate species that are difficult to separate from the active product.
• Processing Complexity: Production requires expression of a prorelaxin precursor that must be cleaved into the A and B chains and then assembled, creating multiple potential kinetic bottlenecks .
• Proteolytic Degradation: The expressed protein or its precursor can be susceptible to degradation by host-cell proteases , especially in the secretion pathway, further reducing the yield.

A successful solution must enhance expression and, critically, ensure the fidelity of the folding process.

Solutions

CD Biosynsis utilizes advanced protein and host engineering to optimize rHRLX production in Pichia pastoris :

Optimization of Pichia pastoris Secretion System

           

We optimize the expression cassette, focusing on strong promoters (AOX}1$) and the alpha-mating factor secretion signal to maximize precursor expression and secretion.

Overexpression of Protein Folding Auxiliary Factors

We co-express folding assistants like protein disulfide isomerase (PDI) and ER chaperones (e.g., Kar}2p$) to enhance correct disulfide bond formation and prevent aggregation.

Genetic Fusion Strategy for Chain Assembly

We design a single chain pro-relaxin construct with optimized cleavage sites, ensuring stoichiometric production of both the A and B chains for efficient downstream assembly.

Host Protease Knockout and Feed Strategy Optimization

We utilize strains with reduced protease activity or optimize the methanol feed rate during fermentation to mitigate product degradation and cell stress.

This systematic approach is focused on overcoming folding complexity to ensure high yield of active rHRLX in the fermentation broth.

Advantages

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

High Active Titer and Yield

Optimized promoters and folding result in a significantly increased concentration of correctly folded rHRLX in the medium.

Improved Folding Fidelity

Auxiliary factor overexpression drastically reduces mispairing of disulfide bonds , maximizing the percentage of active product.

Simplified Downstream Purification

Higher quality secreted product reduces the need to separate complex inactive aggregates or refold inclusion bodies, lowering costs. [Image of Cost Reduction Icon]

Established Industrial Host Platform

Pichia pastoris is a proven, high-density host for industrial protein production, offering a cost advantage over mammalian systems .

High Bioactivity of Final Product

Ensuring correct disulfide bond formation means the final isolated rHRLX has maximal therapeutic activity .

We provide a specialized platform aimed at maximizing the quality and cost-effectiveness of rHRLX biomanufacturing.

Process

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

• Vector Design and Secretion Signal Optimization: Construct the pro-relaxin fusion gene and test various signal sequences for optimal cleavage and secretion efficiency.
• Folding Machinery Co-Expression: Introduce plasmids or integrate genes for overexpressing PDI or other folding chaperones into the Pichia genome.
• Host Strain Engineering: Utilize or generate Pichia strains with knockouts of vacuolar proteases to minimize degradation of the secreted product.
• Fed-Batch Fermentation Optimization: Optimize the fermentation control strategy, including pH and methanol feed profile , to balance growth, induction, and protein stability.
• Product Quality Validation: Analyze the secreted protein via HPLC and Mass Spectrometry to confirm correct disulfide bond formation and measure active titer.
• Result Report Output: Compile a detailed Experimental Report including gene constructs, folding factor expression levels, and final titer/activity data , supporting CMC documentation.

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

Explore the potential for a high-quality, cost-effective rHRLX supply. CD Biosynsis provides customized protein and host engineering solutions:

• Detailed Folding Fidelity and Activity Report , demonstrating the success of disulfide bond correction.
• Consultation on primary recovery and purification strategies optimized for secreted yeast protein.
• Experimental reports include complete raw data on volumetric productivity and product purity profile , essential for scale-up.