Diphtheria Toxin Fusion Protein Production via Periplasmic Targeting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The production of Ontak®, a recombinant protein, is hindered by the formation of protein aggregates and the occurrence of vascular leak syndrome (VLS) in patients, due to its bacterial expression system and inherent toxin properties, leading to purity and safety concerns.
Innovation Solution
Development of DNA expression vectors that include a tox promoter and a mutant tox operator to regulate gene expression, allowing for the production of aggregate-free, monomeric diphtheria toxin fusion proteins in Corynebacterium diphtheria, which are secreted into the culture medium and have reduced VLS side effects by modifying specific amino acid residues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Ontak is produced using E. coli cytoplasmic expression system, then production yield is achieved, but protein aggregates form in the final formulation
Solution Approach 1:
The patent introduces a periplasmic targeting signal peptide as an intermediary element that redirects the Ontak protein from cytoplasmic aggregation-prone environment to the periplasmic space, where proper folding occurs without aggregate formation. This mediator enables the protein to navigate the production system while maintaining purity.
Solution Approach 2:
The patent modifies the cellular location parameter by redirecting protein expression from cytoplasm to periplasm. This parameter change fundamentally alters the folding environment, eliminating aggregate formation while maintaining high production yields through optimized periplasmic expression conditions.
2Reliability
If c-Ontak is used for treatment, then therapeutic effect is achieved, but vascular leak syndrome occurs in patients
Solution Approach 1:
The patent applies local quality modification by making specific amino acid substitutions at particular positions within the Ontak protein structure. These localized changes at critical residues reduce vascular leak syndrome while preserving the overall therapeutic mechanism and efficacy of the protein.
Solution Approach 2:
The patent changes the chemical parameter of the protein by introducing amino acid substitutions that modify the toxin's interaction with cellular targets. These parameter changes reduce harmful vascular leak effects while maintaining the desired cytotoxic activity against cancer cells.
3Productivity
If inclusion bodies are used as protein source, then production efficiency is improved, but denaturation and refolding complexity increases
Solution Approach 1:
The patent performs preliminary action by directing the protein to fold correctly in the periplasm during expression, rather than allowing aggregate formation and requiring subsequent denaturation and refolding steps. This preliminary proper folding eliminates complex downstream processing while maintaining high production efficiency.
Solution Approach 2:
The patent extracts the problematic denaturation-refolding step from the production process by preventing aggregate formation in the first place through periplasmic targeting. This removes the complex and time-consuming denaturation-refolding operations while maintaining efficient protein production.
Data Source
AI summary
The present invention is a DNA expression vector comprising: a toxP; a mutant toxO that blocks Fe-mediated regulation of gene expression; and a DNA sequence encoding a protein, wherein the toxP and the mutant toxO regulate expression of the DNA segment encoding the protein. It is preferred that DNA expression vectors of the present invention include DNA sequences encoding a signal peptide so that a protein expressed is attached to the signal peptide prior to processing. Novel proteins are produced off of the DNA expression vector of the present invention.