Truncated Promoter for Reliable Recombinant Gene Expression
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Solution Overview
Problem
Eukaryotic promoter engineering in biotechnology faces challenges due to unpredictable effects of insertions or deletions in nucleotide sequences, leading to unreliable prediction of essential regulatory elements and high risks of introducing mutations during DNA replication, which complicates the production of recombinant proteins.
Innovation Solution
A truncated promoter is developed, shortened from the 5'-end to 140-610 base pairs, maintaining high expression strength and identity, allowing for the use of shorter synthetic oligonucleotides in PCR reactions and reducing the risk of mutations, while maintaining or exceeding the expression efficiency of non-truncated promoters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If eukaryotic promoter length is reduced to shorten synthetic oligonucleotides for PCR, then the risk of mutations during DNA replication is reduced, but the prediction of essential regulatory elements becomes less reliable
Solution Approach 1:
The patent extracts only the essential regulatory elements from the full-length eukaryotic promoter, creating a truncated version that retains promoter function while removing non-essential sequences. This extraction approach reduces the promoter length to 50-200 base pairs, thereby reducing the risk of mutations during PCR while maintaining reliable gene expression control.
Solution Approach 2:
The patent identifies and preserves specific local regions within the promoter that contain essential regulatory elements such as TATA boxes, CAAT boxes, and GC boxes. By focusing on these specific local quality features rather than the entire promoter sequence, the truncated promoter maintains its essential functions while reducing overall length and complexity.
2Manufacturing precision
If promoter length is shortened, then the risk of introducing mutations during DNA replication is reduced, but the expression strength may be compromised
Solution Approach 1:
The patent optimizes specific parameters of the truncated promoter sequences, including the precise positioning and composition of regulatory elements such as TATA boxes, CAAT boxes, and GC boxes. By carefully adjusting these parameters within the shortened 50-200 base pair sequence, the patent maintains high expression strength comparable to full-length promoters while reducing the overall length to minimize mutation risks during DNA replication.
3Reliability
If full-length eukaryotic promoters are used, then regulatory elements are complete, but the promoter length increases leading to higher mutation risk during PCR
Solution Approach 1:
The patent extracts only the essential regulatory elements from the full-length eukaryotic promoter, creating a truncated version that retains promoter function while removing non-essential sequences. This extraction approach reduces the promoter length to 50-200 base pairs, thereby reducing the risk of mutations during PCR while maintaining reliable gene expression control.
Solution Approach 2:
The patent applies partial action by including only the necessary regulatory elements rather than the complete full-length promoter sequence. The truncated promoter contains 50-200 base pairs with essential regulatory motifs, which is sufficient for reliable gene expression control without the excessive length that would increase mutation risk during DNA replication and PCR operations.
Data Source
AI summary
A promoter or variant thereof has increased relative expression efficiency, in particular a formate dehydrogenase promoter variant has increased relative expression efficiency, and a method provides for recombinant protein production and for increasing the relative expression efficiency of a nucleotide sequence having promoter activity.