CyaA Toxin Refolding via Molecular Confinement
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Solution Overview
Problem
The adenylate cyclase toxin CyaA from Bordetella pertussis tends to aggregate into non-functional multimers due to its hydrophobic character, making it difficult to maintain solubility and stability during purification and storage, which hinders its use in vaccines and immunotherapy, as chaotropic agents like urea are required to prevent aggregation but destabilize other antigens and lead to enhanced multimer formation upon dilution.
Innovation Solution
Refolding of urea-unfolded CyaA under molecular confinement, specifically using size-exclusion chromatography with small particle and pore sizes, in the presence of calcium and post-translational acylation, to favor the formation of monomeric and functional species, which are stable without chaotropic agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If CyaA toxin is purified and stored in the presence of high concentration of chaotropic agent (urea) to prevent aggregation, then solubility is maintained, but other antigens are destabilized and multimer formation is enhanced upon dilution
Solution Approach 1:
The patent introduces molecular confinement as an intermediary mechanism during refolding to prevent multimer formation. By using size-exclusion chromatography with small pore sizes, the toxin molecules are physically constrained during the refolding process, ensuring they fold into monomeric forms without aggregating, thereby eliminating the need for chaotropic agents that destabilize other antigens
Solution Approach 2:
The patent changes the physical parameters of the refolding environment by implementing molecular confinement through specific chromatography conditions. This parameter change allows the toxin to refold into stable monomeric forms in physiological buffers without requiring high concentrations of urea, thus maintaining solubility while preserving functional integrity and avoiding destabilization of other vaccine antigens
2Stability of the object's composition
If CyaA toxin is diluted in physiological buffers to remove chaotropic agents, then storage stability is improved, but multimer formation is enhanced
Solution Approach 1:
The patent applies preliminary molecular confinement during the refolding step before final dilution and storage. By confining the toxin molecules in small-pore chromatography media during refolding, the toxin is pre-formed as stable monomers that resist aggregation even after subsequent dilution in physiological buffers, thus maintaining both storage stability and monomeric purity
Solution Approach 2:
The patent converts the naturally hydrophobic tendency of CyaA toxin (which causes aggregation) into a benefit by using molecular confinement during refolding. The confinement forces the hydrophobic regions to be properly buried within monomeric structures rather than exposing them for intermolecular aggregation, thus transforming the aggregation-prone nature of the toxin into a means of producing stable monomeric forms that maintain purity after dilution
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
The method produces a stable, monomeric, and functionally active CyaA toxin that exhibits higher biological activities and remains non-aggregated during storage, overcoming the limitations of existing methods by maintaining solubility and functionality without denaturing conditions.
Implementation Method 1
Refolding of urea-unfolded CyaA under molecular confinement, specifically using size-exclusion chromatography with small particle and pore sizes
Implementation Method 2
Refolding of urea-unfolded CyaA under molecular confinement, specifically using size-exclusion chromatography with small particle and pore sizes
Implementation Method 3
in the presence of calcium and post-translational acylation, to favor the formation of monomeric and functional species
Implementation Method 4
The acylation region, spanning residues 800 to 1000, contains two post-translational modification sites that are essential for the cytotoxic activities of CyaA
Data Source
Figure 1A~1D
Figure 2A~2F
Figure 3A~3E
AI summary
The present invention discloses a procedure to produce a monomeric and functional form of Bordetella sp, especially B. pertussis, CyaA toxin that can be stably maintained in this functional monodisperse state, even in the absence of any chaotropic agent.