Bacillus subtilis Expression of Antimicrobial Peptide CAD
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Solution Overview
Problem
Current methods for expressing antimicrobial peptide CAD using virus vector systems are cumbersome, difficult to control, and not suitable for mass production, with high production costs and incomplete amidation in yeast, leading to inefficiencies in industrialization and sterilizing activity.
Innovation Solution
A recombinant Bacillus subtilis expression system is developed, utilizing the SUMO protease fusion with antibacterial peptide AD, where the CAD-SUMO fusion products are hydrolyzed to produce active antimicrobial peptide CAD, allowing for high-level expression and secretion without harming the host bacterium, enabling easier and cost-effective large-scale production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If virus vector system is used to express antimicrobial peptide CAD, then expression of CAD genes can be achieved, but the procedure becomes cumbersome and difficult to control
Solution Approach 1:
The patent extracts the CAD gene from the complex virus vector system and expresses it directly in Bacillus subtilis cells using a simplified plasmid-based expression system. This removes the cumbersome virus vector construction and propagation steps while maintaining CAD gene expression capability.
Solution Approach 2:
The patent uses a disposable plasmid vector system instead of persistent virus vectors. The expression plasmid can be easily transformed into Bacillus subtilis and discarded after use, eliminating the need for complex virus vector maintenance and propagation procedures.
2Reliability
If virus vector system is used to express antimicrobial peptide CAD, then CAD genes can be expressed, but mass production becomes difficult and costs increase
Solution Approach 1:
The patent changes the host organism from eukaryotic insect cells infected with virus vectors to prokaryotic Bacillus subtilis cells. This parameter change enables scalable fermentation processes suitable for mass production, as B. subtilis can be cultivated in large volumes under controlled conditions without the complexity of viral propagation.
3Ease of manufacture
If Pichia pastoris is used to express antimicrobial peptide CAD, then secretion and antibacterial activity are achieved, but amidation remains incomplete affecting sterilizing activity
Solution Approach 1:
The patent changes the expression host from Pichia pastoris to Bacillus subtilis, which naturally performs complete amidation of antimicrobial peptides. This parameter change ensures proper post-translational modification and full sterilizing activity while maintaining secretion capability.
4Object-affected harmful factors
If eukaryotic cells are used to express antimicrobial peptide CAD, then no inhibiting effect on cells is observed, but production cycle becomes long and costs increase
Solution Approach 1:
The patent inverts the approach by using prokaryotic B. subtilis instead of eukaryotic cells. Although prokaryotes can have harmful effects on eukaryotic cells, B. subtilis expresses CAD peptides without inhibiting its own growth, and the peptide structure remains stable. This inversion achieves faster production cycles while maintaining peptide functionality.
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 Bacillus subtilis system achieves high protein concentration and strong biological activity of antimicrobial peptide CAD, overcoming the limitations of existing methods with simpler procedures, lower costs, and improved expression yields.
Implementation Method 1
the CAD-SUMO fusion products have no antibacterial activity. There is no harmful effect on the host bacterium. Therefore, the host can express the fusion protein for a long time continuously. The substrate fusion protein needs to be hydrolyzed by the SUMO protease in order to produce the active antimicrobial peptide CAD substrate.
Data Source
AI summary
The present invention relates to a method for expressing antimicrobial peptide CAD by means of a recombinant Bacillus subtilis expression system. The SUMO protease expression operon is first artificially synthesized. The protein expression operon genes of Saccharomyces cerevisiae small ubiquitin-related protein is then fused with the antibacterial peptide AD. The fusion protein is further cloned into the pNF11 plamid to be introduced into Bacillus subtilis, thereby ensuring the induced expression of recombined Bacillus subtilis in shake flasks. The method has the advantages of a simple expression system, large-scale production, low production cost, strong biological activity and no toxic or harmful substance production. Moreover, the method provides a medicine with low price and strong antibacterial capacity for clinic disease prevention and treatment. This invention can also be used as a feedstuff additive.