Meningococcus Promoters for Stable Protein Expression
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Solution Overview
Problem
Current methods for modifying protein expression in meningococci, such as using outer membrane vesicles (OMVs) for vaccination, face challenges in stability and efficiency due to phase variation caused by poly-G sequences in promoters like the parA promoter, and require improved promoters for consistent and high-level expression of desired proteins.
Innovation Solution
Development of modified promoters that lack the wild-type poly-G sequence between the -35 and -10 regions, utilizing a -10 region and a -35 region from meningococcal parA or rRNA gene promoters, with specific spacer sequences and upstream regulatory elements to enhance expression stability and levels of outer membrane proteins in OMVs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the wild-type parA promoter with poly-G sequence is used to drive gene expression, then high-level expression can be achieved, but phase variation occurs causing expression instability
Solution Approach 1:
The invention extracts and removes the problematic poly-G tract sequence from the parA promoter while retaining the essential -35 and -10 regions that drive transcription. This extraction eliminates the source of phase variation (the repetitive G sequences that cause slippage) while preserving the promoter's ability to drive high-level gene expression.
Solution Approach 2:
The invention changes the sequence parameters of the promoter by replacing the poly-G tract with an alternative sequence that has similar transcriptional activation properties but lacks the repetitive structure causing instability. This parameter change maintains expression levels while eliminating phase variation.
2Adaptability or versatility
If gene knockout is performed to remove undesirable genes, then protein composition can be modified, but additional genetic engineering steps are required
Solution Approach 1:
The invention creates a universal promoter system that can control the expression of any gene of interest without requiring gene knockout. By using this modified promoter upstream of target genes, researchers can achieve precise protein composition control through expression regulation alone, eliminating the need for additional knockout steps.
3Productivity
If strong promoters like porA, porB, lgtF, or hpuAB are used to up-regulate protective outer membrane proteins, then expression levels increase, but phase variation and expression instability may occur
Solution Approach 1:
The invention takes the harmful poly-G sequence that causes phase variation and converts it into a benefit by using the well-characterized parA promoter structure while removing only the problematic repetitive element. The resulting promoter maintains strong transcriptional activity but eliminates the instability mechanism, turning a potentially harmful feature into a reliable expression system.
Data Source
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AI summary
New promoters are described to drive transcription in meningococcus e.g. for over-expression of protein antigens for retention in membrane vesicles. Modified porA promoters lack the wild-type poly-G sequence which can cause phase variation. Meningococcal rRNA-coding genes (e.g. for 16S rRNA) can be used to drive transcription of a protein-coding gene. These approaches can be used in combination.