Recombinant Trypsin Production via Chaotropic Solubilization
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Solution Overview
Problem
The efficient manufacture of large quantities of recombinant trypsin for protein pharmaceuticals is hindered by instability in expression systems, activation of trypsinogen by endogenous host cell enzymes, low solubility in bacterial host cells, improper folding, and contaminating chymotryptic activity in animal-derived trypsin preparations, leading to low yields and unwanted cleavage products.
Innovation Solution
A process for producing recombinant trypsin from prokaryote host cells involves solubilizing trypsinogen in a chaotropic agent and a low molecular weight reducing agent without oxidizing agents, followed by refolding at high concentrations and auto-catalytic activation, with subsequent chromatography to remove impurities, enabling high-yield production of pure recombinant trypsin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If trypsinogen is expressed in prokaryote host cells, then large quantities of recombinant trypsin can be produced, but the trypsinogen becomes unstable and activates prematurely by endogenous host cell enzymes
Solution Approach 1:
The patent applies preliminary action by expressing trypsinogen as an inactive precursor that requires specific activation conditions. The trypsinogen is designed with an activation peptide that prevents premature activation during expression and purification, allowing stable accumulation of large quantities before intentional activation occurs in the final step
Solution Approach 2:
The patent utilizes parameter changes by controlling pH, temperature, and the presence of specific activators to prevent premature activation during expression. The activation is triggered only under specific conditions (pH change, presence of enterokinase or autocatalysis at controlled temperatures), allowing separation of production stability from activation timing
2Productivity
If trypsinogen is produced in bacterial host cells, then high yield is achieved, but improper folding and low solubility occur
Solution Approach 1:
The patent uses an intermediary approach by expressing trypsinogen in inclusion bodies (insoluble aggregates) which protect the protein from improper folding in the cytoplasm. The inclusion bodies are then solubilized under controlled conditions with reducing agents to maintain disulfide bond integrity, followed by refolding steps that achieve proper native structure
Solution Approach 2:
The patent applies parameter changes by using chaotropic agents (urea, guanidine hydrochloride) and reducing agents (dithiothreitol, beta-mercaptoethanol) to solubilize inclusion bodies, then gradually removing these agents to enable proper refolding. Temperature control during refolding (10-25°C) prevents aggregation and ensures correct folding
3Power
If animal-derived trypsin is used, then high activity is achieved, but contaminating chymotryptic activity and disease risk are present
Solution Approach 1:
The patent applies copying by creating a recombinant version of animal trypsinogen (bovine, porcine, or human) expressed in bacterial host cells. This recombinant copy eliminates disease risks (BSE, Creutzfeldt-Jakob disease) and contaminating chymotryptic activities present in animal-derived preparations while maintaining the desired tryptic specificity and high enzymatic activity
Solution Approach 2:
The patent extracts only the desired tryptic activity from animal trypsinogen by expressing it recombinantly in bacteria. This eliminates the harmful contaminants (chymotryptic activities, disease agents) that are inherently present in crude animal-derived preparations, achieving purification at the source rather than through post-production cleaning
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 achieves high refold efficiencies and commercially relevant amounts of recombinant trypsin with improved stability and purity, reducing the risk of unwanted cleavage and contamination, thus addressing the challenges of recombinant trypsin production.
Implementation Method 1
providing the recombinant trypsinogen in a solubilization solution comprising a chaotropic agent, a buffer agent, and a low molecular weight reducing agent
Implementation Method 2
infusing the solubilization solution comprising the recombinant trypsinogen over time into a diluent comprising the chaotropic agent, buffer agent, and low molecular weight reducing agent in the absence of a low molecular weight oxidizing agent to provide a refold solution comprising the recombinant trypsinogen at a concentration greater than 1 g/L
Implementation Method 3
incubating the refold solution comprising the recombinant trypsinogen for a time sufficient for the recombinant trypsinogen to refold into a conformation characteristic of native trypsinogen and form the disulfide bonds characteristic of native trypsinogen
Implementation Method 4
a process for producing recombinant trypsin from prokaryote host cells involves solubilizing trypsinogen in a chaotropic agent and a low molecular weight reducing agent without oxidizing agents, followed by refolding at high concentrations and auto-catalytic activation
Data Source
AI summary
A process for producing recombinant trypsin from prokaryote host cells in high yield and high specific activity is described. In particular, a process for producing recombinant trypsin from E. coli is described.