Bacillus subtilis hbFGF Production via Neutral Protease Leader
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Solution Overview
Problem
The production of authentic human basic fibroblast growth factor (hbFGF) is hindered by low yields and high costs, with existing methods often resulting in unauthentic or bioinactive products, making commercially viable production unaffordable.
Innovation Solution
An engineered biological system using a recombinant DNA construct with specific promoters and a neutral protease leader sequence is introduced into Bacillus subtilis to facilitate the expression and secretion of authentic hbFGF, with treatments like lysozyme and Triton X-100 optimizing the secretion process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to produce hbFGF, then the production cost is high, but the yield is low and the product authenticity is compromised
Solution Approach 1:
The patent introduces a neutral protease leader sequence (NPR) as an intermediary element between the promoter and the hbFGF coding sequence. This NPR acts as a mediator that facilitates proper protein processing and secretion while maintaining authenticity. The NPR is cleaved off to produce the authentic mature hbFGF, resolving the contradiction between high yield and product authenticity by providing a controlled processing mechanism.
Solution Approach 2:
The patent employs multiple VegC promoters with different strengths (first VegC promoter and second VegC promoter) to control expression levels at different stages. By adjusting promoter strength parameters and using inducible expression systems, the patent optimizes both yield and authenticity. The use of specific culturing conditions and treatment protocols (lysozyme, Triton X-100) further modifies parameters to enhance secretion of authentic protein.
2Quantity of substance
If large scale production is attempted, then the quantity increases, but the product loses authenticity and bioactivity
Solution Approach 1:
The neutral protease leader sequence serves as a protective intermediary during large-scale production. It guides proper folding and processing of the hbFGF protein even under industrial-scale conditions, ensuring that the final product maintains bioactivity. The NPR sequence is designed to be cleaved precisely, leaving the authentic mature protein with full bioactivity regardless of production scale.
Solution Approach 2:
The engineered Bacillus subtilis system performs self-processing of the hbFGF protein through its native protease system. The bacteria automatically cleave the NPR sequence and secrete the mature authentic hbFGF into the culture medium. This self-service mechanism ensures consistent product quality and bioactivity across different production scales without requiring complex external processing.
3Ease of manufacture
If commercial production is implemented, then the cost decreases, but the yield remains insufficient for commercial viability
Solution Approach 1:
The patent extracts the hbFGF protein from the bacterial cells into the culture medium through secretory expression. By using the NPR leader sequence and optimizing secretion pathways, the patent enables easy extraction of high quantities of authentic hbFGF from the culture supernatant. This approach eliminates the need for complex cell lysis and purification steps, making the process commercially viable with high yield and ease of manufacture.
Solution Approach 2:
The patent implements continuous expression and secretion of hbFGF in Bacillus subtilis cultures. The system maintains continuous production of authentic hbFGF that accumulates in the culture medium, allowing for continuous harvesting. This continuous useful action enables scalable commercial production with consistently high yields and maintains product authenticity throughout the manufacturing process.
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 method achieves a significant increase in hbFGF production, reaching up to 40 mg/l without concentration, and ensures the authenticity and bioactivity of the produced protein, making it more commercially viable.
Implementation Method 1
a first VegC promoter, lacq operator, a second VegC promoter and DNA coding for human basic fibroblast growth factor
Implementation Method 2
introducing said DNA construct in a host for expression of the human basic fibroblast growth factor
Implementation Method 3
the neutral protease leader sequence is positioned between the second VegC promoter and the DNA coding for human basic fibroblast growth factor
Implementation Method 4
the method may comprise a step of, during the production of the authentic human basic fibroblast growth factor, treating the host with lysozyme
Implementation Method 5
the method may comprise a step of treating the host with Triton X-100
Data Source
AI summary
The present invention is concerned with an engineered biological system for production of authentic human basic fibroblast growth factor (hbFGF). The system makes use of a bacterial host and has a recombinant DNA construct with an insert. The insert contains a first VegC promoter, lacq operator, a second VegC promoter and DNA coding for human basic fibroblast growth factor.