Palindromic Operator Protein Expression System
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Solution Overview
Problem
T7-based protein expression systems in microbial expression face challenges such as the need for phage polymerase, which can cause undesirable infections and increase complexity, and require multiple levels of induction, making them difficult to operate and optimize for heterologous protein expression.
Innovation Solution
A perfect palindrome operator sequence-based protein expression system using host RNA polymerase-based promoters like E. coli RNA polymerase, with promoters such as T7A1, T7A2, and lac, and operator sequences like lac, gal, and deo, to provide improved control and inducible expression without phage polymerase, suitable for both prokaryotic and eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If T7-based expression systems are used, then high-level protein expression can be achieved, but the system requires phage polymerase which causes undesirable infections and increases system complexity
Solution Approach 1:
The patent extracts and eliminates the T7 phage polymerase component from the expression system, replacing it with host RNA polymerase. This removes the source of phage particle formation and infection risks while maintaining protein expression capability through host-based transcriptional control mechanisms
Solution Approach 2:
The patent creates a universal expression system that functions in both prokaryotic and eukaryotic cells by using host RNA polymerase instead of phage-specific polymerase. This multi-functional approach allows the same basic expression construct to work across different cell types without requiring phage-specific components
2Productivity
If T7-based expression systems are used, then high-level protein expression can be achieved, but multiple levels of induction are required which makes operation difficult
Solution Approach 1:
The patent segments the control mechanism into distinct promoter and operator elements that can be independently optimized. The promoter provides basal transcriptional activity while the operator provides inducible control, allowing simplified single-stage induction rather than multiple sequential inductions
Solution Approach 2:
The patent changes the operational parameters from phage polymerase induction to host RNA polymerase induction through chemical inducers like IPTG. This parameter change reduces operational complexity by eliminating the need for multiple induction stages while maintaining high-level protein expression
3Productivity
If heterologous protein expression is performed prior to induction, then protein can be produced, but deleterious effects on host cell growth and plasmid stability reduce overall productivity
Solution Approach 1:
The patent performs preliminary action by placing the expression construct in the host cell before induction, allowing the host to prepare transcriptional machinery and metabolic pathways in advance. This enables rapid protein production upon induction while avoiding the toxic effects of immediate high-level expression
Solution Approach 2:
The patent introduces dynamic control through inducible operators that allow the expression system to transition from a low-activity basal state to a high-activity induced state. This dynamic regulation prevents deleterious effects during the lag phase while enabling high productivity during the expression phase
Data Source
AI summary
A perfect palindrome operator sequence-based protein expression system is provided. The expression system comprises a promoter; and a perfect palindrome operator sequence, wherein the promoter is not T7. The expression system is preferably employed for the production of recombinant proteins by fermentation.