Bacillus subtilis Plasmid for Heat Labile Enterotoxin B-Subunit Production
Solution Overview
Problem
Current methods for producing Escherichia coli heat labile enterotoxin B-subunit face challenges such as high production costs, low yield, long production periods, and safety concerns due to endotoxins and the need for methanol as an inducer in conventional expression systems like E. coli, Brevibacillus choshinensis, Saccharomyces cerevisiae, and Pichia pastoris.
Innovation Solution
A plasmid for Bacillus subtilis expression system encoding Escherichia coli heat labile enterotoxin B-subunit, utilizing a secretory signal peptide for protein secretion and inducible expression elements like rpsJ-lacO or veg6-lacO, which allows for efficient production of 100 mg/L to 200 mg/L of recombinant LT-B with reduced production costs and improved safety.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If E. coli system is used for producing recombinant LT-B, then ease of operation is improved, but production cost increases due to insoluble inclusion bodies requiring additional purification steps
Solution Approach 1:
The patent changes the host organism parameter from E. coli to Bacillus subtilis, which naturally secretes recombinant proteins into the culture medium rather than forming inclusion bodies. This parameter change eliminates the need for complex solubility optimization and reduces purification costs while maintaining ease of operation.
2Ease of operation
If E. coli system is used for producing recombinant LT-B, then ease of operation is improved, but safety deteriorates due to endotoxin contamination
Solution Approach 1:
The patent extracts the harmful endotoxin factor by switching from Gram-negative E. coli to Gram-positive Bacillus subtilis. This organism change removes the lipopolysaccharide endotoxin problem entirely, as Gram-positive bacteria produce different cell wall components that do not have the same endotoxin activity, thereby improving safety without sacrificing operational ease.
3Ease of manufacture
If B. choshinensis system is used for producing recombinant LT-B, then protein secretion is improved, but production period increases to 8 days
Solution Approach 1:
The patent changes the bacterial species parameter from B. choshinensis to Bacillus subtilis, which has faster growth kinetics and shorter generation time. This parameter change maintains the beneficial protein secretion capability while reducing the production period from 8 days to a shorter timeframe, improving productivity.
4Ease of manufacture
If P. pastoris system is used for producing recombinant LT-B, then protein secretion and production reliability are improved, but device complexity increases due to methanol inducer requirements
Solution Approach 1:
The patent extracts the complex methanol inducer system by switching to Bacillus subtilis, which can be induced by simpler, safer substances. This removes the need for methanol handling infrastructure and safety protocols, reducing device and process complexity while maintaining reliable protein secretion capability.
5Object-affected harmful factors
If S. cerevisiae system is used for producing recombinant LT-B, then safety is improved with GRAS status, but production yield decreases to about 3 mg/L
Solution Approach 1:
The patent changes the host organism parameter from S. cerevisiae to Bacillus subtilis, which has superior protein expression capabilities while maintaining GRAS status. This parameter change increases production yield from 3 mg/L to significantly higher levels, improving productivity without compromising safety.
Data Source
AI summary
The present invention provides a plasmid, method and kit for producing heat labile enterotoxin B-subunit based on a Bacillus subtilis expression system. By comparing with the conventional method in the art, the present invention has the advantages of high safety, good yield, and simplified process and is therefore favorable for the commercialization object of heat labile enterotoxin B-subunit in the application of vaccine adjuvant.