Recombinant Chymotrypsin Refolding via PEG Redox Mediation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
There is a need for an efficient and inexpensive process to produce recombinant chymotrypsin from prokaryote host cells that can be used to manufacture other protein therapeutics without unwanted cleavage products, as existing methods face challenges in achieving high refold efficiencies and commercially relevant amounts.
Innovation Solution
A process involving the refolding of recombinant chymotrypsinogen from prokaryote host cells, specifically E. coli, using a solubilization solution with chaotropic agents like urea or guanidinium chloride and low molecular weight reducing agents such as cysteine or dithiothreitol, without a low molecular weight oxidizing agent, to achieve high protein concentrations and refold efficiencies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If refolding is performed using conventional methods with low molecular weight redox pairs, then disulfide bond formation can occur, but refold efficiency remains low and protein concentration must be kept low
Solution Approach 1:
The patent changes the chemical parameters of the refolding environment by replacing conventional low molecular weight redox pairs with a high molecular weight polyethylene glycol redox pair system. This parameter change enables refolding at much higher protein concentrations (10-100 fold increase) while maintaining or improving refold efficiency, as the polyethylene glycol system prevents aggregation and allows proper disulfide bond formation even at concentrated conditions
2Productivity
If refolding is performed at high protein concentrations, then commercially relevant amounts can be produced, but aggregation and misfolding increase
Solution Approach 1:
The patent introduces polyethylene glycol as an intermediary substance that mediates the refolding process at high concentrations. The polyethylene glycol redox pair acts as a mediator that facilitates disulfide bond formation while simultaneously preventing protein-protein aggregation through steric effects and excluded volume effects, thereby maintaining folding correctness even at high productivity levels
3Object-generated harmful factors
If low molecular weight reducing agents are used without oxidizing agents, then unwanted cleavage products are reduced, but disulfide bond formation is inhibited
Solution Approach 1:
The patent changes the molecular weight parameter of the redox system from low to high. The polyethylene glycol redox pair, being high molecular weight, provides a different kinetic and thermodynamic profile compared to low molecular weight systems. This allows the reducing agent to protect against unwanted cleavage while the high molecular weight nature prevents excessive reduction of disulfide bonds, and the system still enables proper disulfide bond formation through controlled oxidation
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This process enables the production of recombinant chymotrypsin at commercially relevant amounts with higher refold efficiencies than previously attainable, ensuring safe and consistent manufacturing of protein therapeutics.
Implementation Method 1
a solubilization solution comprising a chaotropic agent, a buffer agent, and a low molecular weight reducing agent
Implementation Method 2
a low molecular weight reducing agent such as cysteine or dithiothreitol
Implementation Method 3
form the disulfide bonds characteristic of native chymotrypsinogen
Data Source
AI summary
A process for refolding recombinant chymotrypsin produced from prokaryote host cells is described. In particular, the present invention provides a process for refolding recombinant chymotrypsin produced from E. coli is described.