Botulinum Toxin Chain Assembly Using GP41.1 Intein Splicing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing botulinum neurotoxin face challenges such as low yield, high production costs, and safety hazards due to the need for cleaving and linking of the heavy and light chains, which are difficult to express in soluble form in E. coli, and require additional processes for disulfide bond formation.
Innovation Solution
A method involving the expression and purification of botulinum toxin fragments using GP41.1 intein and a soluble partner, followed by protein trans-splicing to link the heavy and light chains without a soluble partner, and forming a disulfide bond between them, thereby increasing solubility and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If botulinum toxin is produced using traditional methods requiring cleavage and disulfide bond formation, then the toxin can be obtained, but the production yield decreases and costs increase
Solution Approach 1:
The botulinum toxin is divided into separate heavy chain and light chain components that are expressed independently as fusion proteins with intein tags. These separate components are then purified and reassembled through protein trans-splicing, avoiding the need for proteolytic cleavage and subsequent disulfide bond formation steps required in traditional methods.
Solution Approach 2:
GP41.1 intein serves as an intermediary element that facilitates the assembly of heavy and light chains. The intein tags on separate toxin chains enable their specific pairing and trans-splicing to form the complete active toxin, replacing the need for endogenous proteases and oxidizing systems.
2Productivity
If botulinum toxin is expressed in E. coli, then production can be achieved, but the toxin cannot be expressed in soluble form
Solution Approach 1:
Soluble partner proteins are used as intermediaries to express the botulinum toxin chains in E. coli. The toxin chains are fused to soluble partner tags that enhance their solubility during expression, allowing the toxic components to be produced in a safe, soluble form that can later be purified and reassembled.
Solution Approach 2:
The toxin is segmented into separate chains that are individually expressed with soluble tags. This segmentation allows each component to be optimized for solubility and safety during expression, while the final active toxin is reconstituted through controlled trans-splicing of the intein tags.
3Manufacturing precision
If additional processes are added for disulfide bond formation, then correct toxin structure can be achieved, but the production process becomes more complex
Solution Approach 1:
The GP41.1 intein system enables the toxin chains to self-assemble into the correct disulfide-bonded structure through protein trans-splicing. The intein-mediated reaction automatically forms the native disulfide connections between heavy and light chains without requiring external oxidizing agents or complex folding conditions.
Solution Approach 2:
The invention changes the chemical parameters of the assembly process by using intein trans-splicing instead of traditional disulfide bond formation. This parameter change allows the reaction to proceed under milder, more controlled conditions with higher precision and fewer additional process steps.
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 enables safe, high-yield production of botulinum toxin in a soluble form, simplifying the production process and avoiding the formation of inactive dimers, thus enhancing the toxin's activity and reducing production costs.
Implementation Method 1
screening for a suitable intein, and joining a heavy chain and a light chain of the botulinum toxin to each other using the intein
Implementation Method 2
forming a disulfide bond between them
Data Source
Figure 1
Figure 2(A)~2(B)
Figure 3(A)~3(B)
AI summary
Disclosed is a method of producing botulinum toxin by a recombinant method using a soluble partner and GP41.1 intein. It was found that a heavy chain receptor binding domain (HC) of botulinum toxin with low solubility may be expressed in a soluble form in E. coli by linking a soluble partner. In addition, it was found that the soluble partner, which is generally a protein having a very large molecular weight, is easily removed using GP41.1 intein.