Periplasmic Toxin Expression in E. coli
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Solution Overview
Problem
The production of significant quantities of diphtheria toxins like CRM197 for use in vaccines is hindered by low protein abundance and difficulties in expressing these toxins in E. coli, including periplasmic secretion that leads to decreased host cell viability and increased proteolysis.
Innovation Solution
An improved process for periplasmic expression of bacterial toxins involves growing a culture of bacterial host cells with an expression vector containing specific signal sequences linked to the toxin, inducing expression to produce the toxin periplasmically, and using polynucleotides that encode for these sequences to enhance production and purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If periplasmic secretion is used to express diphtheria toxin in E. coli, then the toxin is produced in its mature form with correct folding, but host cell viability decreases and proteolysis increases
Solution Approach 1:
The patent extracts and removes the toxic diphtheria toxin gene (tox gene) from the Corynebacterium diphtheriae genome, creating an atoxigenic strain that cannot produce the toxic NT toxin. This extraction of the harmful genetic element allows the bacterium to be used as a safe vaccine platform while still eliciting protective immunity, directly resolving the contradiction between producing the toxin for vaccination and maintaining host cell safety
Solution Approach 2:
The patent uses an intermediary approach by employing a recombinant expression system where the diphtheria toxin gene is cloned into an E. coli expression vector under the control of an inducible promoter. This intermediary system allows controlled production of the toxin or toxoid without the risks of using live C. diphtheriae, and enables optimization of expression conditions to balance protein folding quality with cell viability
2Manufacturing precision
If periplasmic secretion is used to express diphtheria toxin in E. coli, then the toxin is produced in its mature form, but proteolytic degradation increases
Solution Approach 1:
The patent applies preliminary action by using protease-deficient E. coli strains (such as BL21(DE3) or its derivatives lacking lon and ompT proteases) for the expression of diphtheria toxin. This pre-selection of protease-deficient host cells prevents proteolytic degradation before the toxin can be harvested, allowing the toxin to accumulate in its mature, correctly folded form in the periplasm without significant degradation
Solution Approach 2:
The patent employs a disposable expression system where E. coli cells are grown in large-scale fermentation, induced to express the toxin, and then harvested and lysed to release the periplasmic contents. The bacterial cells are essentially used once and then disposed of, allowing optimization of expression conditions without concern for long-term cell health or contamination risks
3Productivity
If the tox signal sequence is removed to increase expression, then protein abundance increases, but periplasmic secretion capability is lost
Solution Approach 1:
The patent employs dynamic control of the expression system by using an inducible promoter (such as T7 promoter controlled by IPTG induction) that allows the expression of diphtheria toxin to be turned on only when needed. This dynamic control enables the cells to first grow and accumulate biomass, then upon induction, rapidly produce high levels of toxin or toxoid, achieving both high productivity and controlled periplasmic secretion
Solution Approach 2:
The patent utilizes parameter changes by optimizing various expression conditions including induction temperature, induction timing, IPTG concentration, and culture duration to maximize toxin production. By changing these parameters, the system can achieve high protein abundance while maintaining functional periplasmic secretion, as the toxin is naturally targeted to the periplasm when the signal sequence is present
Data Source
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AI summary
Compositions and methods related to periplasmic expression of a toxin including diphtheria toxin or CRM197 are provided herein.