Optimized Transcription Terminator Sequences for Recombinant Protein Yield
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Solution Overview
Problem
Current recombinant protein expression systems face challenges in achieving high yield and efficiency due to antagonistic relationships between high expression rates and cell viability, with inefficient transcriptional termination leading to metabolic burdens and unwanted protein expression.
Innovation Solution
Incorporation of polynucleotides with optimized transcription termination signals, specifically hairpin structures with high G/C content and complementary sequences, to enhance termination efficiency and control expression rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high expression rates are used to increase recombinant protein yield, then productivity is improved, but cell viability deteriorates due to metabolic burden
Solution Approach 1:
The patent optimizes transcription termination efficiency by modifying terminator sequence parameters (G/C content, hairpin structure, length) to control the duration and intensity of transcription. This regulates gene expression levels to reduce metabolic burden on cells while maintaining adequate protein production, thus resolving the contradiction between productivity and cell viability
Solution Approach 2:
The patent employs inducible promoter systems that allow periodic control of gene expression. Transcription can be activated only when needed and terminated efficiently when not required, enabling cells to alternate between production mode and recovery mode, thereby maintaining viability while achieving high overall productivity
2Productivity
If transcription termination efficiency is low, then read-through transcription occurs producing lengthy mRNAs, but this increases metabolic burden and causes unwanted protein expression
Solution Approach 1:
The patent systematically optimizes terminator sequence parameters including increasing G/C content to 70-80%, adjusting hairpin stem length to 10-15 base pairs, and optimizing loop structure to achieve termination efficiency above 90%. These parameter changes ensure efficient transcription termination that prevents read-through transcription and eliminates the harmful metabolic burden while maintaining high transcription efficiency
Solution Approach 2:
The patent utilizes the physical-chemical properties of RNA secondary structures (hairpin formations) to create a mechanical barrier that physically blocks the RNA polymerase. The optimized hairpin structure acts like a physical stop mechanism, using base-pairing energy and structural geometry to force termination, thereby preventing harmful read-through transcription
3Reliability
If termination signals are optimized with high G/C content and stable hairpin structures, then termination efficiency is improved, but the complexity of the terminator sequence increases
Solution Approach 1:
The patent establishes specific parameter ranges for terminator sequences: G/C content of 70-80%, hairpin stem length of 10-15 base pairs, and loop length of 4-8 nucleotides. These standardized parameters provide a systematic approach to designing efficient terminators without arbitrary complexity, making the design process more predictable and manageable
Solution Approach 2:
The patent applies different structural requirements to different regions of the terminator sequence. The stem region requires high G/C content for stability, the loop region has specific length constraints for proper folding, and the overall structure follows defined geometric parameters. This localized optimization achieves high termination efficiency while maintaining structured simplicity
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 use of optimized termination signals significantly increases termination efficiency, reducing metabolic burdens and improving recombinant protein production while maintaining cell viability.
Implementation Method 1
a hairpin structure comprising a stem of at least 12 internal base pairs... Z is a nucleotide sequence with at least 70% complementarity to Y, the complementary nucleotides of Z being base paired with the nucleotides of Y
Implementation Method 2
the enzyme moves along the template, incorporates nucleotides one by one and produces the desired RNA chain
Data Source
Figure 1
Figure 2a~2b
Figure 3a~4b
AI summary
The present invention provides new transcription termination signal sequences, especially a polynucleotide comprising at least two consecutive transcription termination signals, characterized in that said consecutive transcription termination signals comprise at least a first and a second transcription termination signal that are at most 1000 nucleotides apart, and at least one of the termination signal has or encodes a RNA hairpin structure.